Chapter 2

Review Of

Literature

21

Chapter 2

REVIEW OF LITERATURE

The science of plant tissue culture takes its roots from path breaking research

in botany like discovery of cell followed by propounding of cell theory by Schleiden

(1838) and Schwann (1839). They proposed that a cell is the basic unit of an organism

and capable to regenerate into whole plant if an appropriate environment is given. But

Schleiden and Schwann had no experimental evidence to prove it. The Cell Theory

received much impetus from the famous aphorism of Virchow (1858), ‘‘Omnis cellula

e cellula’’ (All new cells arise from pre-existing cells) and by the very prescient

observation of Vötching (1878) that the whole plant body can be built up from ever so

small fragments of plant organs.

An important approach of tissue culture was discovered by Rechinger (1893)

who tried to determine experimentally the limit of plant divisibility permitting tissue

proliferation. He used isolated buds, slices of roots, stems and other materials. The

explants were placed on sand moistened with tap water. But he did not use nutrients

or aseptic conditions; his culture could scarcely be called tissue culture. However,

Rechinger’s experiments were suggested by a concept related to the tissue culture

principles; thus he was recognized as a true pioneer in this field (Gautheret 1983).

In 1902 Haberlandt, a German physiologist was the first to conduct

experiments designed to demonstrate totipotency of plant cells by culturing isolated

leaf mesophyll cells of Lamium purpureum, glandular hairs of Pulmonaria and cells

from petioles of Eicchornia crassipes on diluted Knop’s (1865) salt solution enriched

with glucose. Unfortunately, he failed largely because of the poor choice of

experimental materials, inadequate nutrients and infection (Vasil and Vasil 1972).

But, he boldly predicted that it should be possible to generate artificial embryos

(somatic embryos) from vegetative cells which encouraged subsequent attempts to

regenerate whole plants from cultured cells. This potential of a cell is known as

‘totipotency’, a term coined by Steward in 1968. Despite lack of success, Haberlandt

made several predictions about the nutrients’ requirement in experimental conditions

which could possibly induce cell division, proliferation and embryo induction.

Haberlandt is thus regarded as ‘father of tissue culture’.

22

Initial progress in plant tissue culture came from the work of Molliard (1921)

in France, Kotte (1922) in Germany, Robbins (1922) in the United States, who

successfully cultured the fragments of embryos and roots. Unfortunately, the growth

of those cultured tissues could not be sustained for long even if they were transferred

to fresh medium. Innovative plant tissue culture techniques progressed rapidly during

the 1930s due to the discovery of natural auxin and vitamin B5

The breakthrough progress came from White (1934) who was the first to

demonstrate continuous culture of excised tomato root-tips on a medium containing

inorganic salts, sucrose and yeast extract (YE). Later, he (1937) replaced YE by

vitamin B namely pyridoxine, thiamine and proved their growth promoting effect.

One of the main thrust in the history of tissue culture is the induction of callus.

Gautheret (1934) is credited with the first successful attempt of callus induction from

cambial cells of some tree species. This was followed by the formation of continuous

callus cultures in carrot and tobacco independently endorsed by Gautheret, White and

Nobécourt in 1939.

which were necessary

for the growth of isolated tissues containing meristem. In 1926, Went discovered the

first plant growth regulator, indole-3-acteic acid (IAA) which is a naturally occurring

member of a class of plant growth regulator (PGR) termed as ‘auxin’.

Adding to the ongoing improvements in the culture media, van Overbeek

(1941) used coconut milk besides usual salts, vitamins and other nutrients for embryo

culture. After 1950, there was an immense advancement in the area of PGR. Skoog

and Tsui (1951) demonstrated continued induction of cell division and bud formation

in tobacco by adenine and high levels of phosphate. This led to further investigations

by Miller et al. (1955) who isolated ‘kinetin’ (Kn), a derivative of adenine (6-furyl

amino purine). They worked further and proposed the concept of hormonal control for

organ formation in 1957. Their experiment on tobacco pith culture showed that the

high concentration of auxin promoted rooting; whereas high kinetin induced bud

formation. Later studies led to the isolation of other naturally occurring as well as

synthetic cytokinins, elucidation of their role in cell division and bud development

and their extensive use in the micropropagation industry related to their suppression

of apical dominance resulting in the development of many axillary shoots.

In early 1960s, the most significant breakthrough in the field of plant tissue

culture was the development of a defined culture medium by Murashige and Skoog

(1962), prepared by increasing the concentration of salts twenty-five times higher than

23

Knop’s solution. Today MS medium has been proved as the most effective and widely

used culture medium for various plant species. The role of tissue culture in plant

genetic engineering was first exemplified by Kanta et al. (1962). They developed a

technique of test tube fertilization which involved growing of excised ovules and

pollen grains in the medium thus overcoming the incompatibility barriers at sexual

level. In 1966, Guha and Maheshwari cultured anthers of Datura and raised embryos

which developed into haploid plants initiating androgenesis.

Recently, tissue culture technology gained unbeatable recognition in plant

science for successful micropropagation and improvement of plant species, leading to

its commercial application. A number of plant species have been micropropagted

around the globe, out of which the review of some important medicinal plants has

been categorized below;

2.1 Organogenesis

Organogenesis is a complex phenomenon involving the de novo formation of

organs (shoots or roots). Shoots can be derived either through pre-existing

meristematic tissues known as ‘axillary shoot formation’ or through differentiation of

non-meristematic tissues known as ‘adventitious shoot formation’. Both these

approaches require synergistic interaction of physical and chemical factors. A

successful plant regeneration protocol requires appropriate choice of explant, age of

the explant, definite media formulation, specific growth regulators, genotypes, energy

source, gelling agent and other physical factors including light regime, temperature

and humidity (Bhojwani and Razdan 1983).

2.1.1 Meristem, shoot tip and nodal segment culture

Meristem culture is based on suppressing the shoot apical dominance by

addition of cytokinins to the growth medium followed by axillary bud sprouting in

multiple shoots. As the cells of apical and axillary bud are uniformly diploid and least

susceptible to genotypic changes, they produce a large number of genetically stable

plants in a short span of time, thus it is regarded as the most common technique for

mass production of useful plant species.

The history of meristem culture began with the first successful shoot tip

culture of Nasturtium (Tropaeolium majus) by Ball in 1946. Since then meristem

24

culture has attracted much attention of the plant scientists. However, demonstration of

practical utilisation of this important technique must be credited to Morel and Martin

(1952) who for the first time produced virus-free Dahlia plants from infected

individual by excising and culturing their shoot tips in vitro. Later, Morel extended

this approach for the production of virus free plants of orchids (Morel 1960). That

was the beginning of tissue culture. Thereafter, in the 1970s developed countries

began commercial exploitation of this technology.

The meristem tip must be small enough to eradicate viruses and other

pathogens, yet large enough to develop into a shoot. In case of meristematic

propagation, elimination of virus particles in explant cells is reached within a short

time. In many cases meristematic cells do not contain virus particles due to absence of

vascular connection with other plant parts. Now, meristem culture is considered as a

unique technique to produce pathogen-free (bacteria, fungi, viruses, viroides and

mycoplasma) plants of many species (Morel and Martin 1955, Walkey 1978,

Bhojwani and Razdan 1983, Biswas et al. 2007). Recently, Banerjee et al. (2010)

used apical meristem for reducing phytoplasma infection in Artemisia roxburghiana.

Plant tissue culture entered in the developing world during the 1980s. It was

earlier used to develop ornamental plants for export. With tree species, the technique

of tissue culture remained confined for many years to laboratory stage and had

generally invited only academic interest. But in most developing countries, the

shortage of biomass and the ever-increasing energy requirements created the need to

explore possibilities of mass propagation of trees by tissue culture. Using this

approach of micropropagation, significant achievements in in vitro cloning has been

made for various herbaceous and woody plants of medicinal, horticultural and

ornamental values (Sharma et al. 2002, Tripathi and Tripathi 2003, da Silva 2003,

Zhou and Wu 2006, Rout et al. 2006, Chaturvedi et al. 2007).

2.1.1.1 Effect of adenine-based cytokinins on shoot regeneration

Cytokinins are plant hormones promoting cell division and differentiation.

Since the discovery of first cytokinin i.e., Kn, a number of chemicals suited to the

definition of cytokinin has grown to include a large array of natural and synthetic

compounds, adenine and phenylurea derivatives. The natural cytokinins are adenine

derivatives and can be classified by their configuration of N6-side chain as isoprenoid

25

or aromatic cytokinins (Fig. 7). Cytokinins with an unsaturated isoprenoid side chain

are the most prevalent, in particular those with a trans-hydroxylated N6-side chain i.e.,

trans-zeatin. However, cis-zeatin and N6-(∆2-isopentenyl) adenine (2-iP) are generally

minor components although exceptions exist (Durand et al. 1994, Emery et al. 1998).

Kn and N6-benzyladenine (BA) are the best known cytokinins with ring substitutions

at N6

Three adenine-based cytokinins viz., BA, Kn and 2-iP have been commonly

used for different approaches of micropropagation; however, zeatin rarely screened

for shoot multiplication. It is now well established that cytokinins are effective only at

optimum concentrations. Higher concentrations are not recommended for shoot

proliferation as callus production interferes with subsequent rooting and

acclimatization. Among all the adenine-based cytokinins tested, BA has been found

the most effective for axillary shoot proliferation, but it is recommended that a range

of concentrations should be tested to optimize shoot production. Ault (2002) reported

that in Hymenoxys acaulis var. glabra, BA at 20 µM induced significantly more

axillary shoots (10.3 shoots per explant) than did other cytokinins. In comparison,

23.3 shoots per node at 5.0 µM BA in Mucuna pruriens (Faisal et al. 2006d), 6.3

shoots per node at 8.87 µM BA in Tinospora cordifolia (Raghu et al. 2006), 12.9

shoots per node at 5.0 µM BA in Ocimum basilicum (Siddique and Anis 2008), 8.6

shoots per shoot tip in Spilanthes mauritiana (Sharma et al. 2009b), 20.7 shoots per

node in Veronica anagallis-aquatica (Shahzad et al. 2011), 14.37 shoots per node at

2.22 µM BA in Ceropegia spiralis (Murthy et al. 2010) have been reported.

-position. In the early years of cytokinin research, only cytokinins with an

isoprenoid side chain were thought to be endogenous compounds; however in the mid

1970s BA derivatives were also identified as natural cytokinins (Horgan et al. 1973 &

1975).

The important role of BA in shoot proliferation has been reported for various

taxa of Asteraceae family, such as Wedelia calendulacea (Emmanuel et al. 2000),

Echinacea purpurea (Koroch et al. 2002), Eclipta alba (Dhaka and Kothari 2005,

Husain and Anis 2006), Stevia rebaundiana (Debnath 2008, Sharma and Shahzad

2011), Centaurea ultreiae (Mallóon et al. 2011) etc. Similarly, the superiority of BA

over other cytokinins has also been reported for various Asclepiads like Gymnema

sylvestre (Komavalli and Rao 2000), Hemidesmus indicus (Sreekumar et al. 2000),

Decalepis hamiltonii (Anitha and Pullaiah 2002), Holostemma ada-kodien (Martin

26

Figure 7. Structural modifications in some adenine and urea-based cytokinins

27

2002), Ceropegia spp. (Beena et al. 2003, Nikam et al. 2008, Murthy et al. 2010,

Chavan et al. 2011), Marsdenia brunoniana (Ugraiah et al. 2010).

Borthakur et al. (2000) reported Kn as the best cytokinin for shoot

proliferation in Eclipta alba and Eupatorium adenophorum. Similarly, Özel et al.

(2006) found Kn as more effective cytokinin for regeneration in Centaurea

tchihatcheffii on optimal concentration of 4.5 mg l-1. While, on 2.5 mg l-1 Kn growth

in regenerants was very slow which improved considerably when the regenerants

were transferred to BA (1.0 mg l-1), NAA (2.0 mg l-1) and glutamic acid (50 mg l-1

In several studies, combination of two cytokinins proved to be advantageous

over single cytokinins treatment for apical and axillary bud sprouting. High frequency

shoot multiplication of Eclipta alba was obtained with MS medium containing BA

combined with Kn or 2-iP. The shoots regenerated on a combination of BA (4.4 µM)

and 2-iP (14.7 µM) grew faster than those initiated in BA and Kn combination

(Baskaran and Jayabalan 2007). In Decalepis hamiltonii, the MS medium composed

of 9.1 µM zeatin, 4.7 µM Kn and 0.6 µM IAA proved to optimal for maximum shoot

regeneration and multiplication (5.4 shoots per shoot tip). Further multiplication and

elongation was achieved on medium containing 2.5 µM 2-iP and 0.3 µM GA

(Giridhar et al. 2005). Whereas, Rani and Raja (2010) reported callus-free multiple

shoot formation in Tylophora indica as a function of BA activity alone, but internode

elongation was dependent on the synergistic effect of GA. Similarly, the shoot buds of

Eclipta alba were multiplied and maintained on BA and GA containing MS medium

(Dhaka and Kothari 2005).

)

combination. While, the promotive role of 2-iP on shoot regeneration has been

reported by Cellarova and Hocariv (2004) in Digitalis purpurea and Sujatha and

Ranjitha Kumari (2007 & 2008) in Artemisia vulgaris.

Generally, inclusion of low concentration of auxin to the cytokinin containing

medium results in high frequency shoot production through axillary or apical buds

and indicates the synergistic effect of a cytokinin and an auxin. The presence of NAA

and BA has increased shoot multiplication in Gymnema sylvestre (Reddy et al. 1998),

Mentha arvensis (Shahzad et al. 1999), Spilanthes mauritiana (Bais et al. 2002),

Santolina canescens (Casado et al. 2002), Clitoria ternatea (Shahzad et al. 2007),

Tylophora indica (Faisal et al. 2007), Gynura procumbens (Keng et al. 2009), Carlina

acaulis (Trejgell et al. 2009) etc. Corral et al. (2011) also investigated that the

addition of NAA with BA significantly improved the shoot proliferation efficiency in

28

Crepis novena as compared to the combination of NAA and Kn. They reported that an

average of 49.77 shoots per axillary bud were produced in 100% of cultures on MS

medium comprised of 0.54 µM NAA and 4.44 µM BA. In contrast, Banerjee et al.

(2010) reported maximum shoot regeneration (38.0 shoots per explant) on the

combination of 13.95 µM Kn and 0.27 µM NAA in Artemisia roxburghiana. They

have advocated that the combination of 8.88 µM BA and 0.27 µM NAA was found to

be more stimulative for further multiplication and elongation during sub-culturing.

For Gymnema sylvestre, Komalavalli and Rao (2000) reported a maximum

number of shoots (57.2) induced from axillary node explants on MS medium

containing BA (1 mg l-1), Kn (0.5 mg l-1), NAA (0.1 mg l-1), malt extract (100 mg l-1)

and citric acid (100 mg l-1). Maximum shoot proliferation on BA and IAA

combination has been reported by Sivaram and Mukundan (2003), Debnath (2008)

and Sharma et al. (2009b). The combination of BA and IAA in addition to additives

like adenine sulphate (ADS), arginine, citric acid and ascorbic acid used to establish

the aseptic cultures of Leptadenia reticulata (Arya et al. 2003). Whereas, Devi and

Srinivasan (2008) found optimal response for micropropagation of Gymnema

sylvestre on MS medium containing 1 mg l-1 BA, 0.5 mg l-1 IAA, 100 mg l-1 vitamins

B2 and 100 mg l-1

Gantait et al. (2010) reported an elite protocol for accelerated quality-cloning

in Gerbera jamesonii using shoot tips in which MS medium supplemented with 0.5

mg l

citric acid using nodal explants. In contrast, Beena et al. (2003)

established a protocol for in vitro propagation of Ceropegia candelabrum through

axillary bud multiplication using 8.87 µM BA in combination with 2.46 µM IBA.

-1 NAA and 1.5 mg l-1 BA promoted earliest axillary bud initiation within 5 day in

91.6% of the inoculants. They achieved very high rate of shoot multiplication (14

shoots per explant) when MS medium was fortified with a relatively higher level of

BA (2 mg l-1) and 60 mg l-1

Many researchers studied the comparative efficiency of different explants for

maximum shoot production. Sivanesan and Jeong (2007) achieved more number of

shoots from nodal segments as compared to shoot tips in Pentanema indicum. They

also revealed the additive effect of ADS when added to the BA and IAA containing

MS medium. Trejgell et al. (2009 & 2010) reported maximum shoot regeneration

form the seedling derived shoot tips in comparison to the hypocotyl, cotyledon and

ADS within 27 day of incubation. According to Gantait

and Mandal (2010), ADS acts as an elicitor or enhancer of growth in synergism with

endogenous and exogenously supplemented PGRs in Anthurium anderanum.

29

root explants of Carlina acaulis and Senecio macrophyllus. Similarly, in Stevia

rebaudiana shoot tips were proved to be the most effective explants for shoot

regeneration over nodal segments and leaf explants when cultured on MS medium

supplemented with 1.0 mg l-1 BA and 0.5 mg l-1

2.1.1.2 Effect of urea-based cytokinins on shoot regeneration

IAA (Anbazhagan et al. 2010).

However, Gonçlaves et al. (2010) reported maximum shoot proliferation from nodal

segments than apical shoot tips in Tuberaria major. The proliferation frequency was

not differed by cytokinin type when nodal segments were used. More than 6 shoots

were obtained on BA and zeatin supplemented MS media. However, a differential

proliferation was noticed for shoot tips depending upon the cytokinins. The difference

in shoot multiplication among different explants in response to exogenous PGR could

be a reflection of probable variation of endogenous PGR level (Yucesan et al. 2007)

or different tissue sensitivity to PGR (Lisowska and Wysokinska 2000).

Diphenylurea (DPU) was the first cytokinin-active phenyl urea identified

(Shantz and Steward 1955). Although, this discovery was linked to the detection of a

compound in liquid coconut endosperm, it was later found to be a contaminant from

prior chemical analysis of DPU. This fortuitous discovery however led to the

synthesis of a number of potent analogues such as forochlorfenuron [1-(2-chloro-4-

pyridyl)-3-phenylurea, CPPU] and thidiazuron [N-phenyl-(1, 2, 3- thidiazol)-5-ylurea

TDZ] (Fig. 7) with cytokinin activity exceeding that of natural cytokinins (Takahashi

et al. 1978, Mok et al. 1982). Synthetic phenylureas are less susceptible to the plants’

degrading enzymes than endogenous cytokinins and can persist in the plant tissues for

long periods of time (Mok and Mok 1985, Mok et al. 1987). Besides, there is no

evidence that any phenylurea cytokinin occurs naturally in plant tissues.

TDZ and CPPU have proven advantageous for micropropagation of a wide

range of recalcitrant plant species that do not respond well to amino puirne because of

their tremendous ability to stimulate shoot proliferation (Huetteman and Preece 1993).

The mode of action of these urea derivatives is still unclear even though it appears

quite sure that they inhibit cytokinin oxidase (CKOx) activity (Hare and van Staden

1994) and thus induce cytokinins accumulation within the cells (Victor et al. 1999).

TDZ mediated response has been reported to be influenced by Ca2++ (Yip and Yang

1986, Hosseini and Rashid 2000). Mundhara and Rashid (2006) and Sharma et al.

30

(2011) reported an enhancement in number of responding explants when transient

Ca2++

Application of TDZ induces a diverse array of culture response in plant

tissues. These range from induction of callus to the formation of somatic embryos.

The activity of TDZ varies widely depending on its concentration, exposure time,

cultured explant and species (Murthy et al. 1998). The concentration at which TDZ is

most effective is 10-1000 times lower than the other PGRs (Huetteman and Preece

1993). Therefore, direct comparison between TDZ and purine-based cytokinins at

equimolar concentrations or at similar durations of the treatment is complicated. Short

duration exposure to TDZ has been proved very effective for morphogenesis (Tulać et

al. 2002). Higher levels, on the other hand, promote callus and somatic embryo

fromation (Huetteaman and Preece 1993, Rida et al. 2001, Fengyen and Han

2002). In most of the studies, continuous or more than critical exposure with TDZ

resulted in stunted or abnormal shoot development. Its deleterious effect has also been

well documented in several plant species (Huetteman and Preece 1993, Faisal et al.

2005, Khurana et al. 2005, Ahmad and Anis 2007).

was provided.

Primarily TDZ was used as a cotton defoliant (Arndt et al. 1976) and later

found to mimic cytokinin like activity that was 20 times more effective in dormancy

breaking (Wang et al. 1986). However, extended research showed that TDZ, unlike

traditional cytokinins is capable of fulfilling both the cytokinin and auxin requirement

of various regenerative responses (Mok et al. 1982, Murthy et al. 1998). The list of

plant species exhibiting morphogenesis in the presence of individual TDZ has

continued to increase over the years, facilitating the improvement of tissue culture

technology (Murthy et al. 1998). For some species, the combination of TDZ and

purine-based cytokinins (usually BA) has been found more effective to induce

morphogenetic response than either TDZ or BA alone as reported by Mohamed-

Yasseen (2002) in Hylocereus undatus.

The exploitation of TDZ for regeneration has been reported vastly superior

over adenine-based cytokinin for a number of woody plant species such as Hydrangea

quercifolia (Ledbetter and Preece 2004), Cassia angustifolia (Siddique and Anis

2007), Pterocarpus marsupium (Husain et al. 2007a) and Vitex negundo (Ahmad and

Anis 2007). Apart from woody plant species, TDZ has also shown a promise role for

regeneration in many other plants (including herbs and shrubs) belonging to diverse

31

groups of families such as Hypericum perforatum (Murch et al. 2006), Bacopa

monniera (Tiwari et al. 2001), Artemisia judaica (Liu et al. 2003), Hordeum vulgare

(Ganeshan et al. 2003), Cineraria maritime (Banerjee et al. 2004), Oryza sativa (Gairi

and Rashid 2004), Hyoscyamus niger (Uranbey 2005), Psoralea corylifolia (Faisal

and Anis 2006).

2.1.2 Leaf culture

Direct regeneration from leaf is another alternative step for clonal propagation

and germplasm conservation. Direct de novo or adventitious shoot regeneration is

most preferred if Agrobacterium-mediated gene transfer is to be achieved and leaf

explants are the best suited for both adventitious shoot formation and Agrobacterium-

mediated gene transfer experiments. Hildebrandt et al. (1946) and Hildebrandt and

Ricker (1947) were the first who cultured the excised leaf tissues of tobacco and

sunflower under aseptic conditions. In a large number of studies, leaf has been proved

as one of the most potent explant for a number of plant species (Misra and Datta

2001, Beegum et al. 2007, Saritha and Naidu 2008, Zheng et al. 2009, Sahai and

Shazad 2010).

Leaf explants have been found to be the most regenerative at their proximal or

petiolar end as compared to leaf margin and mid rib portion. In view of this, high

frequency shoot induction at proximal region may be due to higher accumulation of

PGRs (Rajasekharan et al. 1987). There is a physiological gradient in the leaf explant

from proximal to distal end for de novo regeneration of shoot buds. In some reports,

excised petioles have been found to be more effective than leaf segments to exert

shoot organogenesis. The leaf vein is an extension of xylem and phloem of the stem

through the petiole which is surrounded by one or more layers of parenchymatous

cells, but not well specialized for a particular function yet having the capacity for cell

division. These parenchymatous cells are very sensitive to different growth stimuli,

such as PGRs and environmental conditions; therefore they are easier to be

differentiated to new organs than other cells in tissue culture (Gahan and George

2008).

Multiple shoot regeneration from proximal part of the leaves than distal ends

has been reported in a number of plant species including Tagetes erecta (Misra and

Datta 2001), Anthurium andraeanum (Martin et al. 2003), Euphorbia nivulia (Martin

32

et al. 2005), Spilanthes mauritiana (Sharma et al. 2009b), Lysimachia species (Zheng

et al. 2009). However, Sreedhar et al. (2008) reported direct regeneration of shoot

buds on both side of midrib and rarely from regions of smaller veins, but never from

lamina indicating the presence of vascular cells appear to be crucial for de novo

organogenesis from immature leaf explants of Stevia rebaudiana.

2.1.2.1 Effect of adenine-based cytokinins on shoot regeneration

Among the adenine-based cytokinins, BA was found to be the most effective

to induce direct shoot bud regeneration from the leaf explants in a variety of plant

species. BA alone at 8.87 µM induced 38.0 shoot per leaf explant in Ophiorrhiza

prostrata (Beegum et al. 2007). For Chicorium intybus, BA was found nearly twice

more successful than Kn (Yucesan et al. 2007). Sreedhar et al. (2008) reported that a

combination of BA and Kn was found to be an ideal combination for de novo shoot

regeneration from leaf explants of Stevia rebaudiana. The MS medium containing

8.88 µM BA and 4.65 µM Kn resulted in the formation of highest number of shoots

per explant at the end of 7 week of incubation. Hedayat et al. (2009) evaluated direct

organogenesis from leaf and petiole segments of Tanacetum cinerariifolium and

reported that the leaf segments were highly responsive than petiole cuttings and

produced a maximum shoot regeneration (70%) on MS medium supplemented with

4.0 mg l-1 BA and 0.2 mg l-1 2, 4-D. The highest proliferation rate was observed on

MS medium supplemented with 1.5 mg l-1 BA and 2.0 mg l-1

For Coleus forskohlii, Sahai and Shahzad (2010) evaluated leaf size, position,

orientation and season of collection to select the most regenerative explant condition.

Enhanced shoot production and proliferation has been achieved on medium

containing 2.0 μM BA and 0.1 μM NAA wherein, a highest number of 35.0 shoots per

explant were produced. In the same year, Krishna et al. (2010) provided a rapid in

vitro regeneration protocol using leaf explant of C. forskohlii. They used three

different segments of leaf i.e. proximal, middle and distal and cultured on MS basal

medium supplemented with different cytokinins. Comparison of shoot regeneration

response from different leaf segments at 5.0 mg l

NAA.

-1 BAP showed that the distal end

was comparatively most regenerative as induced 45.0 shoots followed by 23.0 and

21.0 shoots from middle and proximal ends. Further elongation was achieved on MS

medium augmented with 0.1 mg l-1 BA and 0.1 mg l-1 IAA combination. For Populus

33

tremula, Huang and Dai (2011) reported a high frequency of shoot regeneration on

10-20 µM zeatin as compared to other cytokinins. A maximum of 92.6 shoots were

induced from each petiole explant when culture on 20 µM zeatin added to MS

medium, while an average of 60.9 shoots were induced from leaf explants on similar

concentration of zeatin.

The existence of synergistic and additive interaction of auxin and cytokinins

combination involves a complex web of signal interactions such as increased

sensitization, receptivity, feedback inhibition and modulation of gene expression

resulting in variable translation of mRNA population (Cline 1991, Eklof et al. 1997,

Schmulling et al. 1997, Armstrong et al. 2004). In most of the studies, combination of

BA and NAA was found to be most effective for regeneration through leaf explants

such as in Chaememelum species (Echeverrigaray et al. 2000), Phellodendron species

(Azad et al. 2005), Saussurea species (Dhar and Joshi 2005). Through leaf disc

cultures of Sansevieria cylindrica, highest shoot regeneration frequency (80%) and

mean number of shoots per explant (13.5) were obtained in MS medium

supplemented with 10.0 µM BA and 0.1 µM NAA (Anis and Shahzad 2005).

Mohapatra et al. (2008) reported a maximum shoot (8.3 shoots per leaf) and shoot

length (2.1 cm) in 81.6% of cultures of Centella asiatica on MS medium

supplemented with 3.0 mg dm-3 BA and 0.05 mg dm-3 NAA. In Lysimachia

nummularia a maximum of 12.73 shoots per leaf explant were induced in 100% of

cultures on MS medium supplemented with 1.0 mg l-1 BA and 0.1 mg l-1

In contrast to BA and NAA combination, in Tagetes erecta leaf culture highest

shoot regeneration established on BA (13.3 μM) and IAA (17.1 μM) combination

(Vanegas et al. 2002). While in Chicorium intybus, 0.5 mg l

NAA (Zheng

et al. 2009). Similarly, Corral et al. (2011) reported mean number of 2.48 shoots per

explant on 2.22 µM BA and 2.69 µM NAA combination from leaf explant of Crepis

novoana. Superiority of NAA with BA over other auxins might be due to the fact that

NAA has more affinity for easy penetration through plasma membrane even without

active uptake as suggested by Nordstrom et al. (2004).

-1 Kn combined with 0.3

mg l-1 IAA gave optimum response with a mean of 19.7 shoots per lamina explant

(Yucesan et al. 2007) and in Ophiorrhiza prostrata combination of 8.87 µM BA and

2.46 µM IBA yielded maximum number of shoots per leaf explant (76.0) (Beegum et

al. 2007). Whereas, Saritha and Naidu (2008) reported direct organogenesis from

juvenile leaf explants of Spilanthes acmella on the medium augmented with BA and

34

IAA. Similarly, the combination of BA (2.0 mg l-1) and IAA (0.5 mg l-1) produced

maximum number of shoots (32.8) from leaf explants of field grown plants of

Solanum nigrum, whereas from in vitro derived leaf explants maximum number of

shoots (38.0) were obtained on BA (3.0 mg l-1) and IAA (0.5 mg l-1

As an additive, GA also plays a very significant role for the induction of shoot

buds from leaf explants. In this context, Sekioka and Tanaka (1981) were of the

opinion that GA can act as a replacement for auxin in shoot induction and thus a ratio

of cytokinins-GA may be decisive for differentiation in certain plant tissues. GA has

also found conducive for promotion of biomass production and enhanced xylem fibre

length in transgenic aspen (Eriksson et al. 2000). A combination of 14.43 µM GA and

4.44 µM BA in the absence of any auxin induced multiple shoot bud differentiation

from the leaf segments of Tagetes erecta (Misra and Datta 2001).

) combination

(Sridhar and Naidu 2011).

Pre-treatment with low temperature improved the regeneration potential of

plant tissues. The enhancement of plant regeneration by low temperature treatment

was related to the alternation of endogenous auxin-cytokinin balance and redox-state

which played a key role in the plant growth and development (Hou et al. 1997, Merce

et al. 2003, Andersone and Levinah 2005). Guo et al. (2007) reported an efficient

micropropagation system for Saussurea involucrata, an endangered Chinese

medicinal plant through leaf explants. A maximum of 66.0% of shoot regeneration

frequency and 5.2 shoots per explant were achieved when explants cultured on a

medium containing 10.0 µM BA and 2.5 µM NAA. Shoot organogenesis was

improved further when the leaf explants were pre-incubated at low temperature and

80.6% of shoot regeneration frequency was recorded with 9.3 shoots per leaf explant

at 4 °C by 5-day pre-treatment period.

2.1.2.2 Effect of urea-based cytokinins on shoot regeneration

Similar to the meristem culture, TDZ has been successfully exploited for

direct regeneration of shoot buds from leaf explant in a number of plant species

(Feyissa et al. 2005). Like adenine-based cytokinins, TDZ in combination with a

suitable auxin also favoured high frequency of shoot regeneration from leaf explants

(Orlikowska and Dyer 1993). Radhika et al. (2006) reported high frequency of shoot

regeneration and high number of shoots per regenerating leaf explant on a wide range

35

of TDZ and NAA combinations in Carthamus tinctorius. Later, Sujatha and Dinesh

Kumar (2007) compared the efficacy of TDZ plus NAA and BA plus NAA

combination for the leaf explants of eleven Carthamus species. They observed highly

prolific adventitious shoot regeneration on MS medium supplemented with 0.2 mg

dm-3 TDZ and 0.2 mg dm-3 NAA in C. tinctorius whereas 0.2 mg dm-3 TDZ plus 1.0

mg dm-3 NAA was found effective for shoot regeneration in C. arborescens. Zeng et

al. (2008) reported an efficient micropropagation system using leaves as explants for

Tigridiopalma magnifica. Up to 7.6 adventitious buds formed per leaf explant after a

40-day culture on MS medium containing 2.0 mg l-1 BA and 0.1 mg l-1 TDZ. During

30-day subculture, the proliferation rate of adventitious bud in cluster was 5.7 on MS

medium supplemented with 2.0 mg l-1 BA and 0.5 mg l-1

Ma et al. (2011) established an efficient propagation and regeneration system

via direct shoot organogenesis for an endangered species, Metabriggsia ovalifolia.

Among various PGRs tested, 2.5 µM TDZ was found to be the most effective to

induce a maximum of 36.7 shoots per leaf explant. Shoot regeneration capacity was

further enhanced when auxin was added to TDZ. Among a wide range of cytokinin

(Kn, BA and TDZ) and auxin combinations, 5.0 µM TDZ along with 0.5 µM NAA

induced a maximum of 79.1 adventitious shoots from each leaf explant.

NAA.

2.1.3 Cotyledon culture

Cotyledons are a potential source of regeneration because of their year-round

availability, ease of culture initiation and applicability to a number of genotypes

(Burger and Hackett 1982, Baker et al. 1999) and represent a good source not only for

micropropagation studies but also serve as a target tissue for transformation studies

(Franklin et al. 2004). Organogenesis from cotyledons was successfully obtained in

Citrullus lanatus (Chaturvedi and Bhatnagar 2001), Dalbergia sissoo (Singh et al.

2002a), Glycine max (Sairam 2003), Capsicum annuum (Joshi and Kothari 2007),

Pongamia pinnata (Sujatha et al. 2008).

As far as the literature is concerned, single cotyledon explant can produce

multiple shoot buds from the proximal cut ends due to the presence of highly

meristematic cells (Guerra and Handro 1988). Similar results were also reported by

Hisajima (1982) who found that up to 10 million shoots of almond species could be

obtained from a single seed explant within a year after several subcultures. This type

36

of response has been initiated from the seeds of many species, particularly legumes

(Vasanth et al. 2004, Maina et al. 2010).

2.1.3.1 Effect of adenine-based cytokinins on shoot regeneration

Webb et al. (1984) showed that cotyledon age can influence the regeneration

response; with older cotyledons has less ability for direct shoot regeneration than

younger ones. According to Hunter and Burritt (2002), cotyledon age influences the

shoot-forming ability of cotyledon explants. Zhang and Cui (2001) studied the

stimulatory effect of different cytokinins on direct plant regeneration from 5-day old

cotyledon explants in Cucumis sativus. 1.0 mg l-1

Singh et al. (2002a) compared the regeneration potential of semi-mature and

mature cotyledons lacking embryonic axes in Dalbergia sissoo. Shoot buds were

induced from the proximal region of semi-mature cotyledons on MS medium

supplemented with 4.44 μM BA and 0.26 μM NAA. Adventitious shoot bud

formation was also noticed from the mature cotyledons. However, unlike the semi-

mature explants, the mature cotyledons exhibited shoot bud differentiation on MS

medium containing 22.20 μM BA without NAA. Pre-culture of mature cotyledons in

liquid MS medium containing 8.88 μM BA for 48 h improved shoot bud regeneration

up to six-fold.

zeatin had a highest efficiency

(85%) over BA, Kn, TDZ.

Vega et al. (2006) examined regeneration efficacy from three different regions

(proximal, middle and distal) of cotyledon explants in six sun flower inbred lines. A

decreasing regeneration was observed from proximal to distal sections for all inbred

lines. Shoot differentiation depends upon the presence of proximal region of explant

regardless of the genotype. Maximum regeneration frequency (87.1%) was noticed for

N 834 genotype. This was in accordance with other studies in which regenerated

plants were obtained from cotyledons at high level of cytokinins (Joshi and Kothari

2007).

Rashid et al. (2010) assessed the in vitro response of two genotypes of tomato

(Lycopersicon esculentum) viz., Punjab Upma and IPA-3 for direct regeneration from

cotyledon explants. They noticed that direct regeneration was significantly influenced

by the genotype. The MS medium supplemented with Kn (0.5 mg l-1) and BA (0.5 mg

l-1) was found optimum for inducing direct shoot regeneration. At this combination of

37

BA and Kn Punjab Upma exhibited a better response in terms of shoot regeneration

per cent (92.49) and average number of shoots per explant (4.78) in when compared

to IPA-3.

Chaturvedi et al. (2010) compared the regenerative potentiality of cotyledon

explants of some Indigenous varieties of Cucurbits. The 7 day-old seedlings were

used as explant source. They reported direct shoot regeneration for the first time from

cotyledons of Cucurbita pepo under the influence of BA (5.0, 10.0 μM) and 3.0 μM

each of BA and 2-iP. Indirect as well as direct regeneration was observed in Cucumis

melo var. utilissimus; BA alone (5.0, 10.0 μM) supported shoot-bud differentiation

indirectly via callusing while in combination with 2-iP at 1.0 μM each promoted

direct regeneration of shoot-buds in cultures.

2.1.3.2 Effect of urea-based cytokinins on shoot regeneration

Fragmentary reports are available on TDZ mediated direct organogenesis from

cotyledonary leaves. In most of the cases TDZ has been found to induce indirect

organogenesis (Radhika et al. 2006). Murthy et al. (1996) achieved stimulation of

direct organogenesis and somatic embryogenesis from cotyledons of Cicer artietinum

when implanted on BA and TDZ amended MS medium. Multiple shoots formed de

novo without an intermediary callus phase at the cotyledonary notch of the seedlings

within 2 to 3 weeks of culture initiation. TDZ was found to be more effective as

compared to BAP as an inductive signal of regeneration. The TDZ induced multiple

shoot formation at all the concentrations tested (1.0 µM to 10.0 µM), although

maximum morphogenic response was observed at 10.0 µM TDZ. Addition of NAA

alone or in combination with BAP to the MS medium failed to invoke similar

response. When the TDZ supplemented medium was amended with L-proline, the

resultant regenerants were mostly somatic embryos. Histological investigations

confirmed the switch in the regeneration pathway from directly formed adventitious

shoots to embryogenesis (Murthy et al. 1996). High frequency of adventitious shoot

regeneration (33.33%) and the highest number of shoots per explant (6.5) from

cotyledons of Carthamus tinctorius was optimized at 0.5 mg l-1 TDZ and 0.25 mg l-1

IBA containing MS medium (Başalma et al. 2008).

38

2.2 Indirect organogenesis

The undifferentiated mass of profusely dividing cells known as callus and

callus mediated regeneration is termed as indirect organogenesis. In vitro callus can

be induced from various parts of the plants like shoot tip, node, inetrnode, hypocotyl,

cotyledon, root, leaf or floral organs and has multiple uses (Pande et al. 2002,

Khurana et al. 2005, Sahai et al. 2010, Parveen and Shahzad 2011). The induction of

callus growth and subsequent differentiation and organogenesis is accomplished with

the differential application of growth regulators and the controlled environmental

conditions in the culture medium (Tripathi and Tripathi 2003). Explants when

cultured on the appropriate medium, usually with both an auxin and cytokinin, gave

rise to an organized, growing and dividing mass of cells. In culture, callus

proliferation can be maintained more or less indefinitely, provided that the callus is

sub-cultured on the fresh medium periodically. During long-term culture, the culture

may lose the requirement for auxin and/or cytokinins. This process is known as

‘habituation’.

Gao and Bjork (2000) reported callus induction and plant regeneration in

shoot tip explant of Valeriana officinalis with the manipulation of various

combination and concentrations of auxins (IAA, IBA and NAA) and cytokinins (BA

and Kn). Influence of different PGRs on high frequency plant regeneration via leaf

callus was also reported in Coleus forskholii (Reddy et al. 2001). Rehman et al.

(2003) reported regeneration via leaf derived callus of Cichorium intybus on modified

MS medium containing 2.0 mM IAA, 5.0 mM Kn and 1000 mg l-1

Faisal and Anis (2003) reported callus formation from leaf explants of

Tylophora indica with the application of dichlorophenoxy acetic acid (2,4-D) or

trichlorophenoxy acetic acid (2,4,5-T) in which 100% cultures showed callus

induction on MS medium supplemented with 2,4,5-T at high level of 10.0 µM. The

characteristics of calli were also greatly influenced by the concentration of auxins and

casein hydrolysate

(CH) with the production of at least five or more shoots from each callus. Efficient

regeneration system has also been achieved from leaf derived callus in Solanum

laciniatum (Okslar et al. 2002). Koroch et al. (2003) established a protocol for the

induction of adventitious shoots from leaf calli of E. pallida. They reported optimum

shoot regeneration frequency (63%) and number of shoots per explants (2.3 shoots per

explants) on 26.6 µM BA and 0.11 µM NAA containing MS medium.

39

cytokinins. In general, maximum callus induction frequency was observed on a high

level of auxin with low cytokinin level. Nodular callus was initiated from young leaf

segments of Pluchea lanceolata, when cultures on Wood and Braun medium (1961)

containing 2.0% sucrose and 5.0 mg l-1 Kn (Kumar et al. 2004). However,

considerable amount of callus formation was observed with the combination NAA

and BA in Leucaena leucocephala (Maity et al. 2005). The maximum callus induction

frequency from stem callus of Ruta graveolens was observed in a combination of 2, 4-

D and BA (Faisal et al. 2006b). Explants cultured on control medium (PGR-free MS

medium) became necrotic and showed no sign of callus formation. Nandagopal and

Ranjitha Kumari (2006) used ADS for high frequency shoot organogenesis from leaf-

derived callus of Cichorium intybus. They observed highest percentage of callus

induction and multiple shoots proliferation was on MS plus B5

Auxins generally stimulate callus formation, but in some cases phenyl urea

derivative i.e., TDZ was also found to possess callus induction properties. Phippen

and Simon (2000) reported callus and shoot induction in Ocimum basilicum when leaf

explants were placed on MS medium supplemented with 16.8 μM TDZ alone. The

combination of TDZ with an auxin (NAA) greatly influences the callus formation

frequency in leaf explants of Cimicifuga racemosa (Lata et al. 2002). Shahzad et al.

(2006) documented maximum callus formation from mature green cotyledons on 0.6

µM TDZ supplemented MS medium in Acacia sinuata. In Hydrastis canadensis, high

frequency of indirect organogenesis was achieved on 2.5 μM TDZ and 5.0 μM NAA,

however sub-culturing of the parent tissue on BA (5.0 μM) containing medium

maximized the production of shoots (He et al. 2007). Faisal et al. (2005) developed a

protocol for high-frequency shoot regeneration and plant establishment from petiole-

derived callus of Tylophora indica. In this plant, optimal callus was developed from

petiole explants on MS medium supplemented with 10.0 µM 2, 4-D and 2.5 µM TDZ.

Adventitious shoot induction was achieved from the surface of the callus after

transferring onto shoot induction medium. The highest rate (90%) of shoot

multiplication was achieved on MS medium containing 2.5 µM TDZ. For Phyllanthus

amarus, Nitnaware et al. (2011) reported maximum callus induction from leaf explant

on 2.26 µM 2, 4-D and 2.32 µM Kn that exhibited higher shoot regeneration (32.4

shoots per culture) after transfer to MS medium containing TDZ.

medium containing

6.66 μM BA, 2.85 μM IAA and 1.36 μM ADS.

40

2.3 Other factors influencing regeneration

Several factors influence the efficiency of in vitro regeneration such as basal

medium, growth regulators, types of additives, age of explants, age of culture,

photoperiod which have been time-to-time reviewed by various workers (Batra 2001).

2.3.1 Effect of different culture media on shoot regeneration

One of the most important factors governing in vitro growth and

morphogenesis of plant tissues is the composition of the culture medium. The basic

nutrient requirements of cultured plant cells are very similar to those of whole plants.

Several media formulations are used for the majority of all cell and tissue culture

work. These media formulation include those described by Murashige and Skoog (MS

1962), White (1963), Linsmaier and Skoog (1965), Gamborg et al. (B5

The development of culture medium formulations was a result of systematic

trials and experimentations. The MS medium was developed for tobacco, based

primarily on the mineral analysis of tobacco tissue. Previously, it was referred as a

‘high salt’ medium due to its high content of potassium and nitrogen salts. The LS

medium is basically MS medium with respect to its inorganic portion, but only

inostiol and thiamine HCl are retained among the organic components. The B

1968), Nitsch

and Nitsch (NN 1969), Schenk and Hilderbrandt (SH 1972) and Lloyd and McCown

(WPM 1980).

5

medium was devised for soyabean callus culture and has lesser amounts of nitrates

and especially NH4+ than MS. Although, B5

For most of the plant species MS medium was proved to be the best for

micropropagation studies. Whereas, Faisal et al. (2007) examined the effect of

different strengths of MS medium (¼ MS, 1/3 MS, ½ MS and MS), each comprising

2.5 µM BA, 0.5 µM NAA and 100 mg l

was originally developed for the purpose

of obtaining callus or for use with suspension culture but it also works well as a basal

medium for whole plant regeneration. The SH medium was also formulated for callus

culture of monocots and dicots while the White’s medium was designed for tissue

culture of tomato roots. Whereas, the NN medium came in to existence for anther

culture and contains a salt concentration intermediate to that of MS and White’s

media (Beyl 1999).

-1 ascorbic acid for axillary shoot proliferation

41

in Tylophora indica. A sharp decline in shoot proliferation efficiency of the explant

was noticed on gradual reduction in salts’ concentration.

However, Chuenboonngarm et al. (2001) successfully used B5 medium for

micropropagation of Gardenia jasminoides through shoo tip cultures. Similarly,

Nandagopal and Ranjitha Kumari (2006) reported the highest percentage of callus

induction and multiplication shoot proliferation on MS and B5

The comparative influence of different culture media formulations on in vitro

response has been assessed by various groups. In Gymnema sylvestre, Komalavalli

and Rao (2000) found MS medium as the best basal medium for shoot sprouting

(62%), number (3.2) and length (2.2) with little callus formation followed by B

medium supplemented

with 6.66 µM BA and 2.852 µM IAA and 1.360 µM ADS. While, in nodal culture of

Terminalia bellerica a maximum number of shoots per explant (10.6) was obtained on

SH medium, but shoots were stunted and exhibiting yellow leaves which intensified

on subsequent subcultures on the same fresh medium. However, growth of shoots was

better on MS medium (Rathor et al. 2008). Woody plant medium (Lloyd and

McCown 1980) has been reported to be more suitable medium for in vitro

regeneration study of woody tree species. The pivot role of WPM for shoot

proliferation has been reported in Cornus florida (Kaveriappa et al. 1997).

5

Husain et al. (2008) and Husain and Anis (2009) achieved the highest shoot

multiplication as well as shoot length on MS basal medium over half-strength MS,

WPM and B

, SH,

WPM and white’s medium. They noticed that shoot buds sprouted on White’s

medium showed only limited development even if they were maintained for longer

period. Similarly, in vitro propagation of various plants belonging to Asclepiadaceae

has also been shown to have optimum growth in MS medium (Chi Won and John

1985, Pattnaik and Debata 1996, Komalavalli and Rao 1997).

5 basal media in Pterocarpus marsupium and Melia azedarach

respectively. Similar results have also been reported on many woody plant species

including Swartzia madagascariensis, Lagerstromia parviflora and Smilax china

(Berger and Schaffner 1995, Tiwari et al. 2002, Song et al. 2010). Song et al. (2010)

provided a comparative analysis of different culture media formulations (½ MS, MS,

2MS, WPM, B5 and SH) to achieve an efficient micropropagation protocol for Smilax

china. As reported in most of the studied the MS medium exhibited higher growth

than those of others. When three different strengths of MS medium were considered,

½ MS resulted in the highest shoot regeneration over MS and 2MS basal media.

42

On the other hand, Warakagoda and Subasinghe (2009) compared these media

(MS, WPM and B5) for in vitro seed germination of Jatropha curcas. In their study

among three media tested, B5 was the best for seed germination of J. curcus.

Therefore, for culture establishment only B5

Mhatre et al. (2000) used four different nutrient media with various

modifications to determine their role in shoot regeneration in Vitis vinifera. The

following media compositions were chosen for the micropropagation protocol;

was selected.

G16, initiation medium, comprised of NN major and minor salts, LS vitamins,

Fe EDTA, 2% (w/v) sucrose, 10 mg l-1 thiamine HCl, 40.53 mg l-1 ADS, 218.4

mg l-1 monobasic sodium phosphate, 2.25 mg l-1 BAP and 0.09 mg l-1

GM2, multiplication medium, comprised of WPM major and minor salts, B

NAA.

5

vitamins, Fe EDTA, 3% (w/v) sucrose, 2 mg l-1 calcium pantothenate, 168 mg

l-1 monobasic sodium phosphate 0.5 mg l-1 IBA and 2.2 mg l-1

MS2, shoot elongation medium, comprised of MS major and minor salts, MS

vitamins, Fe EDTA, 2% (w/v) sucrose, 0.5 mg l

BAP

-1 Bap and 0.2 mg l-1

GR1, rooting medium (liquid), comprised of half-strength MS major and

minor salts, full strength MS vitamins, Fe EDTA, 1% (w/v) sucrose and 0.1

mg l

IAA

-1

They reported that these modified media (G16 and GM2) resulted in healthy

proliferation, none of the culture exhibited hyperhydricity. For this they suggested

that both G16 and GM2 media contain monobasic sodium phosphate in addition to

BAP and this could be responsible for a synergistic effect of cytokinins and NH

IAA.

4+

In a report of 2004, Nas and Read hypothesized that the composition of

minerals and organic substances in proportions similar to those found in the seed

composition could provide an optimum tissue culture medium for micropropagation

of higher plants. Their hypothesis would help to avoid factorial treatments, labour and

explant requirement for obtaining defined tissue culture medium with optimum

response. Using their hypothesis, they first developed a new tissue culture medium

(Nas Medium, NM 2004) for hybrid hazelnuts (Corylus avellana). Threefold higher

shoot length was observed on NM medium than any other media. Moreover, potential

multiplication rates observed on NM (up to 107%) and WPM (up to 85%) were higher

than those on other media. When the composition of NM was further improved (Nas

ions.

43

and Read medium, NRM) in accordance with their hypothesis, shoot length (up to

three fold) and potential multiplication rate (up to 93%) were further enhanced.

2.3.2 Effect of different carbon sources on shoot regeneration

Carbohydrate compounds, normally found in the sieve-tube exudates of plants

have been positively related with a suitable carbon source used in plant tissue culture

medium (Welander et al. 1989). It is well documented that a specific carbohydrate

may have different effects on morphogenesis in vitro. A carbohydrate, generally

sucrose, is an indispensible ingredient of all culture media, as the photosynthetic

ability of cultured tissue is limited because of low irradiance and limited gas exchange

(Kozai 1991). It is also required as an osmotic agent (Thorpe 1985). Being easily

translocable and resistant to enzymatic degradation due to non-reducing nature,

sucrose is the most effective of choice among various carbohydrates for of plant tissue

culture studies (Pontis 1978). But, now it is well established that carbohydrate

requirements may show differences according to the species (Thompson and Thorpe

1987).

A concentration of 20 to 40 g l-1

Although sucrose is the most widely used carbohydrate in tissue culture, some

reports indicate that it may cause hypoxia and ethanol accumulation due to fast

metabolism and result in a significant decrease in osmotic potential of the medium

(Neto and Otoni 2003). These conditions could in turn interfere with the nutrient

uptake process. This interference would most likely result in the failure of absorption

of diffusion or diffusion of some important elements. In such a critical situation, some

reducing sugar like mono- or disaccharides and sugar alcohols such as glucose,

sucrose (a disaccharide made up of glucose

and fructose) is the most often used carbon or energy source, since this sugar is also

synthesized and transported naturally by the plant. Whereas, Murashige and Skoog

(1962) stated that the use of 3% sucrose is better than 2 or 4%. The sugar

concentration chosen is dependent on the type and age of the explant in culture. To

justify this fact, Gürel and Gülsen (1998) investigated the requirement of sucrose

concentration during three successive stages, namely initiation, transplantation and

multiplication for Amygdalus communis cultures. Comparatively higher concentration

of sucrose (5 and 6%) was required during initiation and transplantation stages as

compared to multiplication phase (3 and 4%).

44

fructose, sorbitol and maltose may also be used to find an alternative carbon source

(Nicoloso et al. 2003, Mosaleeyanon et al. 2004, Skrebsky et al. 2004, Pati et al.

2006, Rodrigues et al. 2006, Bandeira et al. 2007, Luo et al. 2009, Dobránszki and da

Silva 2010, Mohamed and Alsadon 2010).

The response of in vitro cultures to different carbon sources added to the

medium has been compared for a number of plant species. Among three carbon

sources (glucose, fructose and sucrose), sucrose proved to be the best for shoot

regeneration in Pentanema indicum (Sivanesan and Jeong 2007). Similar result was

obtained in Artemisia vulgaris (Sujatha and Ranjitha Kumari 2008). In fact, sucrose

has been commonly used as a carbon source in tissue culture media (Fuentes et al.

2005). This is due to its efficient uptake across the plasma membrane (Borkowska and

Szezebra 1991).

Both sucrose and glucose gave a similar rate of proliferation in sour cherry

(Borkowska and Szezebra 1991), Bixa orellana (Neto and Otoni 2003). While,

Debnath (2005) reported the best response at 20 g l-1

Sorbitol, a polyol that occurs abundantly in plant species, is a good carbon

source for Malus species and Prunus persica tissue culture (Chong and Taper 1972,

Coffin et al. 1976). The promotive effect of sorbitol on shoot multiplication rather

than sucrose has also been reported for some plant species of Rosaceae (Pua and

Chong 1984, Kadota et al. 2001, Ahmad et al. 2007).

sucrose in terms of explant

response and shoot developing potential, although glucose supported shoot growth

equally well or better than sucrose depending upon cultivar type of Vaccinium vitis-

idaea. But, carbohydrate concentration had a little effect on shoot vigour. Abou-

Rayya et al. (2010) found glucose as the most effective carbon source for stimulating

the production of shoots, fresh weight and shoot length followed by sucrose and

fructose. In Solanum nigrum, Siridhar and Naidu (2011) also reported the highest

number of shoots (24.0) on 4% fructose, but maximum shoot length (11.0 cm) was

observed on 4% sucrose. The results obtained are in line with the earlier observation

in Mulbery (Vijaya Chitra and Padmaja 2001), where addition of fructose instead of

sucrose in the multiplication medium increases the shoot number and also growth of

shoots.

Energy source has also found to enhance the alkaloid content along with

optimum morphogenesis. In Nepeta rtanjensis, Misic et al. (2005) noticed significant

enhancement in shoot growth and nepetalactone accumulation on glucose. Besides,

45

they also determined the effect of different concentrations of carbohydrates in culture

media on the internal carbohydrate status for the same plant species.

2.3.3 Effect of different pH on shoot regeneration

Scragg (1993) reported that at low pH, cells release H+ to the extracellular

environment affecting the absorption of nutrients, especially the NH4+ while at high

pH, cells release OH- ions thus the absorption of NO3- is adversely affected. Based on

the current report, it is hypothesized that the inhibitory effect of pH on shoot height is

likely to be due to reduced uptake of NH4+ and NO3

-

Martins et al. (2011) studied the influence of low pH (4.5, 5.0 and 5.75) on in

vitro growth and biochemical parameters ((lipid peroxidation, proline and

carbohydrate content, antioxidant enzymes activities and total soluble protein) of

Plantago almogravensis and P. algarbiensis. It was observed that medium pH did not

affect in vitro proliferation and rooting. Interestingly, cultures of both species modify

the initial pH value to the same final value. Results have shown that the lowest pH

tested induced an increase in the level of lipid peroxidation in roots of both species

and in shoots of P. algarbiensis, indicating plasma membrane damage. An

accumulation of carbohydrates was observed in roots of P. almogravensis cultured at

pH 4.5 and 5.0. Based on the results obtained it was concluded that Plantago species

are apt to grow in vitro in medium with pH values much lower than the usually used

in tissue culture, which is in agreement with the fact that both species colonize acid

soils.

at low and high pH respectively.

Every species requires an optimum pH for shoot regeneration and their subsequent

proliferation. In most of the studies optimum regeneration was achieved at 5.8 pH

(Sahai et al. 2010, Shahzad et al. 2011). However, Bhatia and Ashwath (2005) and

Naik et al. (2010) suggested the requirement of acidic pH for maximum biomass

production of Lycopersicon esculentum (5.5 pH) and Bacopa monniera (4.5 pH)

respectively.

2.4 In vitro rooting of microshoots

Rooting is an important step in micropropagation studies (Moncousin 1991).

Although, a number of plants root spontaneously in culture (some monocotyledons

and other herbaceous species), shoots of most species multiplied in vitro lack a root

46

system (Yeoman 1987). Rooting can be achieved either by transferring the

microshoots to medium lacking cytokinins with or without a rooting hormone or by

treating the shoots as conventional cutting after removal from sterile culture medium.

There is a great variation between species in the ease with which cultured shoots can

be rooted and systematic trials are often needed to find the most effective conditions

for rooting. All cytokinins inhibit rooting and BA (which is widely used for shoot

multiplication) does so strongly, even after transfer to cytokinins-free medium

(George and Sherrington 1984). The use of Kn or 2-iP in place of BA in the final

stage of multiplication often improves subsequent rooting (e.g. in Pinus as reported by

Webb and Street 1977). On the other hand, MS basal medium devoid of PGR has

been found to induce in vitro rooting in some plant species (Cristina et al. 1990,

Saxena et al. 1998, Faisal and Anis 2003, Ray and Bhattacharya 2008). The ease of

root formation on auxin free medium may be due to the availability of endogenous

auxin in the regenerated shoots (Minocha 1987).

The concentration of rooting hormone (generally auxin) is often required to

provide sufficient stimulus to initiate roots while preventing the excessive formation

of callus. Root elongation may be inhibited by the levels of auxin required to initiate

roots and the use of IAA which rapidly breaks down in cultured tissues is a useful

way of overcoming this problem without having to provide a second rooting medium.

The requirement of IAA for best rooting has also been reported in Artemisia vulgaris

(Sujatha and Ranjitha Kumari 2007), Stevia rebaudiana (Ahmed et al. 2007,

Anbazhagan et al. 2010), Gerbera jamesonii (Gantait et al. 2010) etc.

Many species require the stronger axuin i.e., IBA or NAA to stimulate root

fromation. Among the auxins tested, IBA was found to be the most suitable for in

vitro rooting (Sreekumar et al. 2000, Faisal and Anis 2002, Liu et al. 2003, Sivaram

and Mukundan 2003, Anis and Shahzad 2005, Feyissa et al. 2005, Faisal et al. 2005a,

Faisal et al. 2006c, Faisal et al. 2007). Half-strength MS with IBA was proved to the

best for in vitro root induction in Saussurea obvallata (Dhar and Joshi 2005), Centella

asiatica (Mohapatra et al. 2008) and Spilanthes acmella (Saritha and Naidu 2008).

However, NAA usually give rise to short and thick roots which may have the

advantage of being better able to withstand accidental damage during planting out

(Lane 1979). The stimulatory effect of NAA on root formation has been reported in

many medicinal plants like Tagetes erecta (Misra and Datta 2001), Carthamus

47

tinctorious (Radhika et al. 2006), Trichosanthes dioica (Malek et al. 2007),

Lysimachia species (Zheng et al. 2009)

Sometimes combination of two different auxins or axuin with cytokinin found

to induce optimum rooting response. The combination of IBA and IAA was found to

induce best rooting in Ficus religiosa (Siwach and Gill 2011). Regenerated shoots of

Anthurium andraeanum were best rooted on half-strength MS medium with 0.54 µM

NAA and 0.93 µM Kn (Martin et al. 2003). Similarly, Beegum et al. (2007) reported

best rooting in Ophiorrhiza prostrata when shoots were cultured on 10.74 µM NAA

and 2.32 µM Kn containing half-strength MS medium.

Different phenolic compounds like, phloroglucinol (PG), chlorengenic acid

(CA) and salicyclic acid (SA) also facilitate in vitro rooting in recalcitrant species and

PG was found to be a promotive phenolic compound for root induction in

Pterocarpus marsupium (Anis et al. 2005, Husain et al. 2007a). However,

Sakhanokho and Kelley (2009) observed that SA in combination with NaCl had a

beneficial effect on root formation along with shoot multiplication and plant survival

in Hibiscus moscheutos.

Auxin promotes ethylene production that inhibits adventitious root formation

in some species like pea cuttings (Nordstrom and Eliasson 1993) and Prunus avium

shoot cultures (Biondi et al. 1990). Ma et al. (1998) demonstrated that the use of

ethylene inhibitors such as AgNO3 and CoCl2 may promote root formation in shoot

cultures of apple. Effect of AgNO3 on root fromation was also examined by Bais et

al. (2000) for Decalepis hamiltonii. They reported best rooting response with the

application of 40.0 µM AgNO3

2.5 Synseed production

. However, Reddy et al. (2002) used triacontanol

(TRIA) for in vitro rooting in D. hamiltonii.

Since the formulation of the concept of synseed by Murashige (1977), a

number of studies have been undertaken in this area of plant biotechnology. The first

report on synseed development was published by Kitto and Janick (1982) who

produced desiccated carrot synseeds by coating the multiple somatic embryos in a

water-soluble resin, polyoxyethyelene glycol (Polyox). Later on, Janick et al. (1993)

extended this technology for encapsulating a mixture of carrot somatic embryos and

embryogenic calli using Polyox. Redenbaugh et al. (1984) was the first to develop

48

hydrogel encapsulation technique for somatic embryos of alfalfa. Since then,

encapsulation in hydrogel remains to be the most studied strategy of synseed

production (Ara et al. 2000, Rai et al. 2009).

A number of coating agents such as agar, sodium alginate, potassium alginate,

sodium pectate carrageenan, sodium alginate with carboxymethyl cellulose, gelatin,

gelrite, guargum, tragacanth gum etc. have been tested as hydrogels (Ara et al. 2000,

Rai et al. 2009). Among these, sodium alginate has been frequently selected because

of its moderate viscosity, low spin ability of solution, low toxicity, quick gellation,

low cost and bio-compatibility characteristics (Swamy et al. 2009). Moreover, sodium

alginate and calcium salt has been reported as the best combination since the ions are

non-damaging, easy to use, have a low-price and provide an easy germination of

encapsulated propagules.

The encapsulation matrix composition is one of the important factors

significantly affecting the re-growth performance of encapsulated tissue. For effective

re-growth, the requirements of definite ingredients of hydrogel matrix (inorganic,

organic, PGRs, carbohydrate etc.) are species specific. Generally, the addition of

nutrients to the gel matrix results in improved re-growth performances (Chand and

Singh 2004, Sundararaj et al. 2010, Ahmad and Anis 2010).

Additionally, synseeds are reported to be highly susceptible to bacterial,

fungal and other infections in the greenhouse (Vij and Kaur 1994). To reduce

microbial contamination, various antimicrobial agents such as bavistin (Pattnaik et al.

1995), vitrofural G-1 (Nieves et al. 2003), plant preservative medium (PPM) (Micheli

2002) could be added to the gel matrix. However, such chemicals induce impairment

in convertibility that could be successfully alleviated by adding PGRs in gel matrix. In

agreement to this, additive effect of PPM and thidiazuron (TDZ) on overall plantlet

development was observed by Lata et al. (2009) in Cannabis sativa synseeds.

Previously, the synseed production was limited mostly to those plants in which

somatic embryogenesis had been reported, but embryogenesis was not well

documented for most of the plant species. In response to this, the possibility of using

non-embryogenic vegetative propagules such as shoot tips, nodal segments,

organogenic or embryogenic calli etc. has been explored as a suitable alternative to

somatic embryos (Ara et al. 2000, Danso and Ford-Llyod 2003, Rai et al. 2008, Faisal

and Anis 2007, Sharma et al. 2009a & b, West and Preece 2009, Ahmad and Anis

2010, Ozudogru et al. 2011).

49

With the use of such non-embryogenic plant propagules, synseed technology

has been widely exploited to a range of plant species such as Morus spp. (Pattnaik et

al. 1995, Pattnaik and Chand 2000), Eucalyptus grandis (Watt et al. 2000), Adhatoda

vasica (Anand and Bansal 2002), Dalbergia sissoo (Chand and Singh 2004), Ananas

comosus (Gangopadhyay et al. 2005), Chonemorpha grandiflora (Nishitha et al.

2006), Punica granatum (Naik and Chand 2006), Cannabis sativa (Lata et al. 2009),

Spilanthes mauritiana (Sharma et al. 2009b), Zingiber officinale (Sundararaj et al.

2010), Vitex negundo (Ahmad and Anis 2010) etc.

Amongst various vegetative propagules, nodal segments are most suitable for

encapsulation studies as they posses pre-existing axillary meristem, however in vitro

root induction is a major obstacle encountered in case of recalcitrant woody plant

species. It is assumed that encapsulation inhibits the oxygen supply to explants and

suppresses root induction (Piccioni 1997). Thus, to induce rooting different

approaches have been exploited for root induction in synseeds.

Piccioni (1997) suggested a method of incubating the explants in dark for root

primordia induction and thereafter addition of PGRs in the gel matrix for higher

conversion from synseeds. Pre-treatment of explants either with cytokinin or auxin

was also suggested for improved synseed conversion frequency (Pattnaik et al. 1995,

Soneji et al. 2002, Chand and Singh 2004, Germanà et al. 2011). Pinker and Abdel-

Rahman (2005) emphasized that the addition of IAA to the gel matrix (prepared in

modified MS) exhibited 100% root formation in encapsulated nodal segments of

Dendraanthema × grandiflora. Nishitha et al. (2006) suggested the addition of silver

nitrate (AgNO3

Addition of growth regulators to the germination medium eliminates the

requirement of an additional in vitro root induction experiment prior to

acclimatization. Ahmad and Anis (2010) found that the addition of Kn and NAA to

MS basal medium improved the germination frequency of synseeds and induced a

mean of 2.8 roots per synseed of Vitex negundo. Similar response has also been

recorded previously for Pimpinella pruatjan (Roostika et al. 2006) and Tylophora

indica (Faisal and Anis 2007). On the other hand, Gangopadhyay et al. (2005) devised

a two step method to achieve maximum synseed conversion into complete plantlets in

Ananus comosus; firstly, the microshoots were retrieved from synseeds and in the

) along with IBA to enhance the conversion frequency in synseeds of

Chonemorpha grandiflora.

50

second step, these microshoots were rooted in liquid medium (supplemented with IBA

and Kn) supported with Luffa-sponge.

Although a large number of plants can be produced in tissue culture through

direct or indirect embryogenesis and organogenesis, but their delivery is cumbersome.

Direct sowing of the synseeds in the soil or other type of substrates helps in escaping

acclimatization procedure. The technique provides an ideal delivery system enabling

easy flexibility in handling and transport of propagules as compared to large parcels

of seedlings. Thus, germination of synseeds on nutrient free substrate is a prerequisite

for sowing under non-sterile condition.

In this context, Mandal et al. (2000) suggested that the successful conversion

of synseeds into plantlets on simple planting substrate such as sand/soil/soilrite/vermi-

compost is necessary for their use in commercial-scale propagation. Still successful

germination of encapsulated tissues on various planting substrates has been reported

only for a few plant species either in a controlled culture room environment or

greenhouse conditions. The major limiting factor for reduced germination is the low-

nutrient availability. Therefore, it is necessary to build up a nutrient reservoir for the

encapsulated plant tissue, either endogenously or exogenously. Kavyashree et al.

(2006) exogenously supplied half-strength LS nutrients in horticultural grade soilrite

mix (peat: perlite: vermiculite 1:1:1) for ex vitro germination of mulberry synseeds

with healthy shoot and root systems. Similarly, Lata et al. (2009) reported 100%

conversion of synseeds on 1:1 potting mix-fertilome with coco natural growth

medium, moistened with full-strength MS medium with 3% sucrose and 0.5% PPM in

Cannabis sativa.

Synseed technology also acts as a tool of germplasm exchange and short term

conservation for rare and endangered plant species. For this purpose synseed storage

is a critical factor which determines their successful germination after transportation

abroad. Therefore, appropriate storage conditions and definite storage period are

prerequisites to maintain viability during exchange of germplasm for successful

commercialization of synseed technology. For short to medium-term storage, the aim

is to increase the interval between subculture by reducing growth. In this respect,

various strategies have been applied for slow growth maintenance of cultures.

Low temperature and light intensity induce modifications in the physiology of

stored explants, such as reduced respiration, water loss, wilting and ethylene

production, thus allowing the storage of cultures from several months to years without

51

the necessity of transferring to fresh medium (Ozudogru et al. 2010). The temperature

requirement for optimum viability varies from plant to plant. Generally, 4 ºC

temperature is found to be most suitable for synseeds storage (Saiprasad and Polisetty

2003, Kavyashree et al. 2006, Faisal et al. 2007, Singh et al. 2007, Pintos et al. 2008,

Sharma et al. 2009a & b, Ikhlaq et al. 2010, Tabassum et al. 2010). Gangopadhyay et

al. (2005) stored the synseeds of Ananas comosus in different racks of a refrigerator

with a range of temperature (4, 8, 12 and 16 ºC) for 60 days. Among the four

temperature regimes, the beads stored at 8 ºC showed maximum germination

frequency when allowed to re-grow again on nutrient media.

Ray and Bhattacharya (2010) optimized best storage environment for Eclipta

alba synseeds by changing in vitro physicochemical conditions. They extended

storage duration up to 12 weeks by decreasing the sucrose concentration in the

alginate matrix from 3 to 1 or 2%. Adriani et al. (2000) has also reported the

pronounced effect of sucrose on re-growth ability of synseed and suggested that the

sucrose availability can be a limiting factor in conversion ability of Actinidia

synseeds.

2.6 Acclimatization of plantlets

The ultimate success of micropropagation depends on the ability to transfer

and re-establish vigorously growing plants from in vitro to green house conditions.

This involves acclimatization or hardening-off plantlets to conditions of significantly

lower relative humidity and higher light intensity. Although, micropropagation has

been extensively used for the rapid multiplication of various plant species and

considerable efforts have been directed to optimize the conditions for in vitro stages

of micropropagation, but the process of acclimatization of tissue culture raised plants

to the natural environment has not been fully studied (Hazarika 2003).

Micropropagation is often restricted due to high percentage of plantlets’ death during

ex vitro transplantation (Pospisilova et al. 1999). The acclimatization of

micropropagated plants remains a critical stage; in the first week after transfer to ex

vitro conditions, plantlets cope with the different stresses and have to adapt to the

external environmental conditions (Aragon et al. 2005). In fact, microparopagated

plants are difficult to transplant due to following of two primary reasons:

52

i) A heterotrophic mode of nutrition

ii) Poor control of water loss (Kane 2000).

A number of researches have been conducted to solve various problems

related to acclimatization such as relative humidity and indicate that high humidity

increase the survival percentage of micropropagated plants (Kozai 1991). A

composite of anatomical and physiological features, characteristic of in vitro plant

produced in 100% relative humidity, contributes to the limited capacity of

microprapagted plants to regulate water loss immediately following transplanting.

These features include no epicuticular wax formation, poor cuticle development,

poorly differentiated mesophyll, poor connection between shoots and roots and

improper function of stomata resulted in excessive water loss and poor photosynthetic

capacity in ex vitro acclimatized plants (Ziv 1991, Kane 2000, Chen et al. 2006).

Leaf surface covering agents, such as glycerol, paraffin and grease also

promoted ex vitro survival of herbaceous species, but have not been evaluated over a

long-term or examined on woody species (Selvapandiyan et al. 1988). Several growth

retardants which reduce damage due to wilting without deleterious side effect have

been suggested to improve ex vitro survival of regenerants. Absiccic acid (ABA) is

considered as a growth retardant which may alleviate ‘transplantation shock’ and

speed up acclimatization of tobacco plantlets under ex vitro conditions (Pospisilova et

al. 1998). Ray and Bhattacharya (2008) established an efficient and simple protocol

for in vitro propagataion of Eclipta alba including successful transplantation by

priming the plantlets with a growth retardant, chlorocholine chloride (CCC) for the

first time. Among various concentrations of CCC, 6.33 µM was found most effective

for inducing certain beneficial changes. In 30 day-old treated shoots, they observed an

increased number of roots, elevation in chlorophyll content and plant biomass. They

reported that the arrested undesirable shoot elongation made the plants sturdy and

more suitable for acclimatization. The primed plants exhibited 100% survival

frequency as compared to control plants (84%). Priming of micropropagated

propagules has already been recommended for obtaining better acclimatized plants

(Nowak and Shulaev 2003, Hazarika 2003). The concept of priming the tissue culture

raised plants to improve acclimatization is based on the fact that certain chemicals

effectively pre-sensitize cellular metabolism of plants (Nowak and Shulaev 2003) and

increase the adaptive ability of in vitro cultures (Conrath et al. 2002, Nowak and

Pruski 2004).