Chapter 2 Review of Literature -...

Chapter 2

Review

of

Literature

15

Chapter 2

REVIEW OF LITERATURE

2.1 A brief history of plant tissue culture

The foundation of plant tissue culture and modern biotechnology can be drawn

back to the “Cell Theory” of Schleiden (1838) and Schwann (1839) which

recognised the cell as the primary unit of all living organisms. This theory holds

that the cell is the primary unit of structure and function in an organism and

therefore capable of autonomy. The concept of ‘totipotency’ itself is inherent in

the cell theory, which forms the basis of modern biology. The cell theory received

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

cellula’ (All cells arise from a cell), and by the very prescient observation of

Vöchting (1878) that the whole plant body can be built up from ever so small

fragments of plant organs. However, no sustained attempts were made to test

the validity of these observations until the beginning of the 20th century

(Gautheret 1985).

The great German botanist Gottlieb Haberlandt (1902), recognised as the father

of plant tissue culture, was the first to conduct experiments designed to

demonstrate totipotency of plant cells by culturing isolated leaf cells of different

plant species like palisade cells from leaves of Lamium purpureum, glandular

hairs of Pulmonaria and pith cells from petioles of Eicchornia crassipes etc. on

Knop’s salt solution (1865) enriched with glucose. In his cultures, cells increased

in size, accumulated starch but failed to divide. He failed largely because of the

poor choice of experimental materials; insufficient nutrients and infection (Vasil

and Vasil 1972). Nevertheless, he boldly predicted that it should be possible to

regenerate artificial embryos (somatic embryos) from vegetative cells, which

encouraged subsequent attempts to regenerate whole plants from cultured cells.

After Haberlandt’s failure Hanning (1904) choose embryogenic tissue to culture

and successfully grew them to maturity on mineral salts and sugar solution. For

the next thirty years (up to 1934) there was very little further progress under cell

culture research. Within this period an innovative approach to tissue culture

using root and stem tips was reported by Kotte (1922) in Germany and Robbins

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(1922) in the United States. Further, Robbins and Maneval (1924) reported some

improvement in root growth but the first successful report of continuously

growing cultures of tomato root tips was made by White in (1934).

The discovery of the naturally occurring auxin indole-3-acetic acid (IAA) by Fritz

Went in 1926 and its beneficial effects on plant growth (Went 1928; Kögl et al.

1934 and Thinmann 1935), soon led to its assimilation in plant nutrient media

(White 1934 and Gautheret 1985). Several years later, in 1939, three

researchers independently used the newly isolated plant growth regulator, IAA

(auxin), to establish callus cultures with the potential for indefinite growth. These

researchers were RJ Gautheret (Gautheret 1939) and P Nobecourt (Nobecourt

1939) from France working with carrot cells and PR White (White 1939) from the

United States, working with tobacco cultures. PR White and A Braun (White and

Braun 1941) found that cells isolated from tobacco infected with crown gall would

show cell division and growth in cultures without the addition of auxin. Proving

one of the predictions of Haberlandt true, in 1941 Van Overbeek and co-workers

demonstrated for the first time the stimulatory effect of coconut milk on embryo

development and callus formation in Datura (Van Overbeek et al. 1941).

Initially, the explants containing meristematic cells were used to obtain unlimited

growth of plant tissues. Continued cell division and bud formation were soon

obtained when tobacco pith tissues that contained mature and differentiated cells

were cultured on nutrient media containing adenine and high levels of phosphate

(Skoog and Tsui 1951). However, cell divisions occurred only when the explant

included vascular tissue (Jablonski and Skoog 1954). A variety of plant extracts

including coconut milk were supplemented to the nutrient medium in an attempt

to replace vascular tissues and to identify the factors responsible for their

beneficial effect. Among these, yeast extract was found to be most effective and

its active component was shown to have purine like properties. This finding led

to the addition of DNA to the medium which greatly enhanced the cell division

activity (Vasil 1959). These investigations resulted in the isolation of ‘kinetin’

from old samples of herring sperm DNA (Miller et al. 1955) and the

understanding of the shoot morphogenesis in plants (Skoog and Miller 1957).

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Later studies led to the isolation of naturally occurring as well as many synthetic

cytokinins. Since then tissue culture studies revolutionized the improvement in

plant species and conservation. The dream of Haberlandt for the cultivation of

somatic embryos comes true when the first report on somatic embryogenesis

appeared in carrot tissue by Reinert (1958) and Steward et al. (1958).

Besides PGRs, scientists tried to improve culture media by differing essentiality

in mineral content. In this direction Murashige and Skoog (1962) prepared a

medium by increasing the concentration of some salts by twenty five times

higher than Knop’s solution (1865). The principal novel features of the new

medium (MS medium) were the very high levels of inorganic constituents,

chelated iron in order to make it more stable and available during the life of

cultures, and a mixture of four vitamins and myo-inositol. Even today MS

medium is the most widely used culture medium and has immense commercial

application in tissue culture.

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

which developed into haploid plants initiating androgenesis (Guha and

Maheshwari 1964; 1966), which like many great advances in science was a

chance discovery (Guha-Mukherjee 1999). The technology was further defined

and improved by the work of Nitsch and Nitsch (1969). Vasil and Hildebrandt

(1965; 1967) were first to regenerate plantlets from colonies of isolated cells of

hybrid N. glutinosa x N. tabaccum. In 1966, the classical work of Steward on

induction of somatic embryos from free cells in carrot suspension cultures

brought an important breakthrough by finally demonstrating totipotency to

somatic cells, thereby, validating the ideas of Haberlandt. Kohlenbach (1966)

successfully cultured mature mesophyll cells from Macleaya cordata, the tissue

obtained from these cells subsequently differentiated somatic embryos. Until the

mid - 1970’s hormonal manipulation in the culture medium remained the main

approach to achieve plant regeneration from cultured cells and it proved very

successful with many species. However, some very important plants such as

cereals and legumes did not respond favourably to this strategy and were,

therefore, declared recalcitrant (Bhojwani et al. 1977). In 1972, Saunders and

Bingham reported that different cultivars of alfalfa varied considerably in their

regeneration potential under a culture regime. More detailed study by Bingham

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and his associates (Bingham et al. 1975 and Reisch and Bingham 1980)

demonstrated that regeneration in tissue cultures is a genetically controlled

phenomenon.

In 1974, Murashige defined three major stages of the micropropagation

technique. These are:

Stage I: Establishment of aseptic cultures.

Stage II: Multiplication and propagation of propagules.

Stage III: Re-establishment in natural environment.

Subsequently, Debergh and Maene (1981) introduced an additional stage that is

Stage 0: care and preparation of stock plant. This is because of the increased

recognition of the importance of the genetic make-up of the plant from which

explants made. Additionally Murashige’s Stage III is further divided into Stage III:

rooting in microshoots and Stage IV: acclimatization of the rooted plantlets.

Stage IV is the most important and crucial stage of micropropagation as plants

slowly acclimatizes to the external environment of lower relative humidity

(Preece and Sutter 1991). However, Stage III and IV can be combined by rooting

microshoots under ex vitro conditions, where rooting and acclimatization took

place simultaneously, as in Murashige’s original Stage III.

Today, tissue culture technique has been widely accepted as a tool for

biotechnology for vegetative propagation of plants of agriculture, horticulture and

forestry importance (Chu and Kurtz 1989 and Dave and Purohit 2002). It allows

rapid and large scale multiplication of selected plant species under controlled

conditions. Micropropagation of various plant species, including many medicinal

plants has been reported. This chapter reviews the achievements and advances

in the application of tissue culture for the in vitro regeneration of medicinal plants

from various explants.

2.2 Micropropagation or in vitro propagation

It is referred to the aseptic culture of cells, tissues, organs and their components

under defined physical and chemical conditions in vitro (Thorpe 2007). It is also

known as ‘micropropagation’ in scientific technology. The technique of

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micropropagation is based on the concept of totipotency as proposed by

Haberlandt (1902), every cell of the plant body is totipotent i.e. capable of giving

rise to a new plant under controlled conditions. Callus mediated organogenesis

and regeneration through somatic embryogenesis are the usual mode of

regeneration by tissue culture. There are three ways or strategies by which

micropropagation can be achieved:

i) Axillary bud breaking – direct regeneration

ii) Production of adventitious buds directly/indirectly via callus -

indirect organogenesis

iii) Somatic embryogenesis directly/indirectly on explants

The use of plant tissue culture technology for the vegetative propagation of

plants is the most widely used application. Micropropagation has many

advantages over conventional method of vegetative propagation, which suffer

from several limitations (Debnath et al. 2006). The most outstanding merits

offered by tissue culture technique over conventional methods are:

1) In a relatively short time and space large number of plants can be

produced, starting from single explant.

2) Unlike the conventional methods of plant propagation,

micropropagation of even temperate species may be carried out

throughout the year.

3) Regenerated plants are generally free from bacterial and fungal

diseases.

4) Virus eradication and maintenance of plants in a virus free state are

also readily achieved in tissue culture.

5) The multiplication rate is greatly increased.

6) It also permits the production of pathogen free material.

2.3 Tissue culture studies in some medicinally important Cassia spp.

Some of the important medicinal species of genus Cassia are C. alata, C.

angustifolia, C. auriculata, C. obtusifolia, C. fistula, C. occidentalis, C. siamea, C.

sophera and C. tora. Various regeneration protocols utilizing different strategies

of micropropagation have been developed for these medicinal plant species

which are summarized in table 3 and have been reviewed by Parveen and

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Shahzad (2012). A brief account of the micropropagation protocols developed for

important Cassia spp. is given below:

2.3.1 Shoot induction and multiplication

2.3.1.1 C. angustifolia Vahl.

This is a highly valuable medicinal species of Cassia, in which micropropagation

studies have conducted using various explants. Agrawal and Sardar (2003)

obtained only 2.4 shoots/explant from cotyledonary nodes of aseptic seedlings of

senna on MS medium supplemented with 1.0 µM BA. However, Siddique and

Anis (2007a) reported the maximum 17.6 shoots from CN explants when

pretreated with 1.0 µM TDZ for 4 weeks and then subsequently transferred to

MS basal medium. Furthermore, Siddique and Anis (2007b) improved the shoot

regeneration efficiency through nodal segments with the production of maximum

21.7 shoots/explant, when the pretreated explants on MS + TDZ (5.0 µM) + NAA

(1.0 µM) for four weeks, were transferred on MS basal medium. Indirect

organogenesis using different explants viz. leaflet, cotyledon, petiole and root

has also been reported in C. angustifolia through induction of regenerative callus

on various hormonal supplements. Agrawal and Sardar (2006) reported

regeneration through leaflets and cotyledons derived callus on MS + BA (1.0 µM)

+ 2,4-D (1.0 µM).

Siddique et al. (2010) reported shoot regeneration through petiole derived callus

induced on MS medium supplemented with 2,4-D (5.0 µM) + TDZ (2.5 µM).

Optimal response with the production of a maximum of 12.5 shoots was

achieved on MS + TDZ (5.0 µM) + IAA (1.5 µM) with an average shoot length of

4.3 cm. However, single TDZ (5.0 µM) treatment was lesser effective and yielded

only 8.5 shoots/explant. Besides leaflet, cotyledon and petiole explants another

explant, root, had been tried by Parveen and Shahzad (2011) for the induction of

regenerative calli and it was found that root explant significantly proved to be the

best explant for the induction of organogenic calli in C. angustifolia. Root

segments (1-2 cm long) taken from 30 days old aseptic seedlings were cultured

on MS medium supplemented with different cytokinins (BA, Kn and TDZ) at

various concentrations. The dark green or brown regenerative calli were induced

on the MS medium containing TDZ (1.0 µM). This regenerative callus was found

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to be suitable for shoot induction when transferred to the MS medium containing

different concentrations (1.0, 2.5 and 5.0 µM) of cytokinins (BA, Kn and TDZ).

Among three cytokinins tested, BA at 2.5 µM produced maximum of 24.5 shoots

per explants after 6 weeks of culture. The synergistic effect of cytokinin-auxin

combination further enhanced the rate of shoot buds formation from the callus.

When the optimal concentration of BA (2.5 µM) was tested with different

concentrations of two auxins (IAA & NAA), a further increase in the number of

shoot buds per culture was achieved. The medium comprised of MS + BA (2.5

µM) + NAA (0.6 µM) proved to be optimal with the production of a maximum of

35.6 shoots/explant having average shoot length of 5.4 cm.

Parveen et al. 2012 reported enhanced shoot organogenesis in C. angustifolia

via callus production through 14 days old cotyledon explants. Callus was

induced at various concentrations of 2,4-D and 2,4,5-T. The organogenic callus

induced at 5.0 µM 2,4-D, proved to be the best for shoot regeneration when

transferred on to MS medium containing cytokinins and auxins singly or in

combination. Maximum shoot differentiation with the production of 23.2 ± 1.4

shoots/explant having shoot length of 5.0 ± 0.3 cm was obtained at BA (5.0 µM)

+ NAA (0.4 µM).

Somatic embryogenesis in C. angustifolia was reported by Agrawal & Sardar

(2007) using immature cotyledons dissected from semi mature seeds and

inoculated on MS medium containing different auxins (2,4-D, NAA or IAA) alone

or in combination with cytokinins such as BA, Kn or 2-iP. A maximum of 75%

cotyledons differentiated into somatic embryos on 2,4-D, however, such embryos

were failed to form complete plantlets. Addition of BA along with 2,4-D improved

this problem. Optimum response was obtained at 2,4-D (5.0 µM) + BA (2.5 µM)

where an average of 5.36 somatic embryos were formed along with 2.0

shoots/culture. Some of the embryos showed precocious germination on the

same medium. However, a few of them developed into complete plants if

transferred to half strength MS basal containing 2% sucrose. Cytokinins alone

did not induce somatic embryogenesis but formed multiple shoots. BA at 5.0 µM

proved optimum for recurrently inducing shoots in the competent callus with a

maximum of 12.0 shoots and an average shoot length of 2.2 cm. Types of auxin

and its interaction with cytokinin significantly influenced somatic embryogenesis.

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Table 3. List of medicinally important Cassia spp. propagated through

tissue culture

Species Explants Best Treatment

Response References

C. alata CN - - Indirect Organogenesis

Fett-Neto et al. 2000

C. angustifolia CN, NS, ST

MS + BA (1.0 µM) 2.4 shoots/CN explant Direct Regeneration

Agrawal & Sardar 2003

C. angustifolia L, C MS + 2,4-D (1.0 µM) + BA (1.0 µM)

12.0 shoots/explant Indirect Organogenesis


C. angustifolia C MS + 2,4-D (10.0 µM) + BA (2.5 µM)

12.0 shoots/explant Somatic Embryogenesis


C. angustifolia NS MS + TDZ (5.0 µM) + IAA (1.0 µM)

21.7 shoots/explant Direct Regeneration

Siddique & Anis 2007a

C. angustifolia CN MS + TDZ (1.0 µM) 17.6 shoots/explant Direct Regeneration

Siddique & Anis 2007b

C. angustifolia P MS + 2,4-D (5.0 µM) + TDZ (2.5 µM)


Siddique et al. 2010

C. angustifolia R MS + TDZ (1.0 µM) 35.6 shoots/explant Indirect Organogenesis

Parveen & Shahzad 2011

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A: Anther; C: Cotyledon; CN: Cotyledonary node; E: Embryo; L: Leaf; NS: Nodal

segment; P: Petiole; R: Root; S: Stem; ST: Shoot tip

C. angustifolia C MS + 2,4-D (5.0 µM) 23.2 shoots/explant Indirect Organogenesis

Parveen et al. 2012

C. auriculata CN MS + BA (1.0 mg/l) + NAA (1.0 mg/l) + AdS (25 mg/l) + ascorbic acid (20 mg/l) + glutamine (150 mg/l)

>16.0 shoots/explant Direct regeneration

Negi et al. 2011

C. obtusifolia ST

MS + 2,4-D (2.0 mg/l) + Kn (0.2 mg/l)


Hasan et al. 2008

C. fistula

E P, S

- B5 + BA (1.0 mg/l) + IAA (0.5 mg/l)

- Indirect Organogenesis

Bajaj et al. 1988 Gharyal & Maheshwari 1990

C. siamea

A P, S CN

2,4-D (2.0 mg/l) + Kn (0.5 mg/l) + CW B5 + BA (1.0 mg/l) + IAA (0.5 mg/l) MS + BA (1.0 µM)

- Callus induction - Indirect Organogenesis 12.2 shoots/explant Direct Regeneration

Gharyal et al. 1983 Gharyal & Maheshwari 1990 Parveen et al. 2010

C. sophera CN MS + TDZ (2.5 µM) 14.9 shoots/explant Direct Regeneration

Parveen & Shahzad 2010

C. tora NS MS + BA (2.2 µM) 1.5 shoots/explant Direct Regeneration

Quraishi et al. 2011

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2.3.1.2 C. auriculata L.

Negi et al. (2011) developed a micropropagation protocol for C. auriculata using

CN explants and also performed anatomical comparison between in vivo and in

vitro developed shoots and roots. Explants containing cotyledonary node along

with cotyledonary leaves and portion of hypocotyl were cultured on MS media

supplemented with different growth regulators in varying concentrations. They

found more than 16.0 shoots/explant in MS medium supplemented with BA (1.0

mg/l) + NAA (1.0 mg/l) + AdS (25 mg/l) + ascorbic acid (20 mg/l) + glutamine

(150 mg/l). Moderate green to brown, hard and nodular callus was observed

originating from cotyledonary leaf margins in MS medium supplemented with BA

(3.0 mg/l) + 2,4-D (1.0 mg/l).

2.3.1.3 C. obtusifolia L.

The in vitro propagation of this valuable medicinal plant from shoot tips was

reported for the first time by Hasan et al. (2008). Different concentrations and

combinations of auxin and cytokinin were used in full strength of MS medium to

obtain callus, shoot regeneration and root proliferation. The highest percentage

of callus (96.6%) was observed on MS medium supplemented with 2.0 mg/l 2,4-

D. A maximum of 5.0 shoots/explant was produced on MS medium comprised of

2.0 mg/l 2.4-D + 0.2 mg/l Kn.

2.3.1.4 C. alata L.

There is a single report on the micropropagation of C. alata (Fett-Neto et al.

2000), where CN explant was used for the induction of morphogenic calli in

83.0% cultures.

2.3.1.5 C. siamea Lam.

Gharyal et al. (1983) reported callus formation from the cultured anthers of C.

siamea. Anthers were split open after 1 to 2 weeks of inoculation on B5 medium

supplemented with coconut water (CW) (15%) + 2,4-D (2.0 mg/l) + Kn (0.5 mg/l)

and produced callus mass. Microscopic examination of the anthers cultured at

the late uninucleate or early bi-celled stages, after 7-14 days of culture, revealed

many multicellular structures at various stages of development, thus indicating

the pollen origin of callus. Gharyal and Maheshwari (1990) also reported the

formation of callus from stem and petiole explant of two important Cassia spp.

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viz: C. fistula and C. siamea. Regenerative calli were induced on B5 medium

supplemented with BA (1.0 mg/l) and IAA (0.5 mg/l), although green

meristemoids were observed from both types of explants but shoots developed

only from stem explants in both the species.

Parveen et al. (2010) for the first time developed an in vitro protocol for the direct

shoot regeneration of C. siamea using CN explants taken from aseptically grown

seedlings and cultured on MS medium supplemented with different cytokinins

(BA, Kn and TDZ) and auxins (IAA, IBA and NAA) either alone or in combination.

Among three cytokinins tested, BA at 1.0 µM produced a maximum of 8.2 shoots

per explants in 80% cultures. The regeneration frequency further enhanced with

the application of auxin along with optimal BA concentration. The highest

number of shoots (12.2 shoots/explant) were obtained at MS + BA (1.0 µM) +

NAA (0.5 µM) with 90% regeneration percentage.

2.3.1.6 C. sophera Linn.

Parveen and Shahzad (2010) for the first time reported the development of a

regeneration protocol for this highly valuable medicinal plant by culturing CN

explant on TDZ supplemented MS medium. Explants were collected from 21

days old in vitro raised seedlings and incubated under controlled condition on

different concentrations of TDZ. TDZ at 2.5 µM proved to be optimal for the

production of maximum number (6.7) of shoots/explant. To avoid adverse effects

of prolonged exposure of TDZ in long term establishment, the cultures were

transferred to TDZ free MS medium fortified with various concentrations of BA

for multiplication, proliferation and elongation of induced shoots. Emergence of

new shoot buds and multiplication continued up to second subculture passage

and maximum number of 14.9 shoots/explant were obtained on MS + BA (1.0

µM).

2.3.1.7 C. tora L.

Micropropagation of this medicinal legume via nodal bud culture method has

been first time reported by Quraishi et al. (2011). Nodal explants taken from field

grown plants were used to initiate in vitro culture with 90% shoot bud induction

producing a maximum of 1.5 ± 0.1 shoots/explant on MS medium supplemented

with 2.2 µM BA. Heavy black leaching was observed in the initiation medium

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from cut ends of the explants, which was effectively checked by incorporation of

an absorbent (25 µM PVP-40) and an antioxidant (476 µM citric acid).

2.3.2 Rooting and acclimatization

In C. angustifolia rooting has been achieved using different strategies of rooting

by different workers. Agrawal and Sardar (2003) reported 80% rooting in C.

angustifolia, with the production of 3-5 roots within 20 days on half strength MS

medium supplemented with 10.0 µM NAA. Rooted plantlets were taken out of

the medium after 1 month, dipped in 0.1% bavistin for half an hour before their

transfer to soilrite. Maintained there for 1 week for in vitro hardening and

subsequently transferred to soil for further acclimatization. However, in other

reports on the same plant species, 95% rooting with 5.4 ± 0.41 roots/shoot

(Agrawal and Sardar 2006) and 91.6% rooting with 5.12 0.58 roots/shoot

(Agrawal and Sardar 2007) were reported on half strength MS medium

containing 10.0 µM IBA. Acclimatization in both these reports was done by

adopting the same procedure described by Agrawal and Sardar (2003). Contrary

to these reports, Siddique and Anis (2007b) achieved only 29-52% rooting with

3.6 ± 0.29 roots/shoot having root length of 3.9 ± 0.40 cm in C. angustifolia using

a two-step rooting procedure by giving a pulse treatment with 60 µM IBA and 1%

activated charcoal in MS medium for 1 week and subsequently transferring the

shootlets to half strength MS liquid medium without IBA and activated charcoal.

The rooted plantlets were hardened off in sterile soilrite for 4 weeks and

eventually established in natural soil. The two-step rooting procedure was again

tried by Siddique et al. (2010) for the production of maximum 3.3 ± 0.25

roots/shoot having root length of 3.1 ± 0.23 cm in 50.5 ± 3.23% microshoots by

giving a pulse treatment with 10 µM IBA for 2 weeks and subsequently

transferring the microshoots to MS liquid medium without IBA. In a recent report

by Parveen et al. (2012), rooting was best induced (82.0%) on half strength MS

medium containing only 1.0 µM IBA along with 5.0 µM PG with the production of

4.8 ± 0.4 roots/shoot having root length of 4.3 ± 0.5 cm after 4 weeks.

Regenerated plantlets with well-developed root system were transferred to

plastic pots containing sterilized soilrite and hardened off under culture

conditions for 4 weeks, after that transferred to earthen pots containing sterilized

soilrite and maintained in green house with 85% survival rate.

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Ex vitro root induction in C. angustifolia was achieved through pulse treatment

with IBA (200 µM) for half an hour followed by their transfer to sterile soilrite

(Siddique and Anis 2007a and Parveen and Shahzad 2011). Ex vitro rooting

decreased the micropropagation cost and the time from laboratory to field

conditions as rooting and acclimatization took place simultaneously. After 6

weeks, the regenerated plantlets were successfully transferred to earthen pots

containing sterilized soil and manure (1:1) and maintained in green house with

90% survival rate (Parveen and Shahzad 2011).

In C. auriculata (Negi et al. 2011) rooting was induced from the cut end of the

hypocotyls in MS medium supplemented with IBA (1.0 mg/l) or NAA (1.0 mg/l).

While, the use of 2.0 mg/l NAA was significant for the production of the highest

5.0 roots/shoot in 80% cultures of C. obtusifolia (Hasan et al. 2008). After 35

days well-rooted plantlets of C. obtusifolia were transferred in plastic pots

containing sterile sand, soil and farmyard manure in the ratio of 1:1:1. After

proper acclimatization, the plantlets were transplanted in the natural condition

with 70% survival.

In Cassia alata (Fett-Neto et al. 2000) rooting was best achieved on IBA

supplemented medium and the regenerated plantlets with well-developed root

system were successfully transferred to soil.

In C. siamea, successful in vitro rooting was induced from cut end of the

microshoots on half strength MS containing 2.5 µM IBA with the production of

3.60 ± 0.24 roots/shoot having root length of 7.88 ± 0.28 cm in 84% microshoots

(Parveen et al. 2010). Plantlets with well-developed root and shoot system were

hardened off inside the growth room in sterile soilrite for 4 weeks. After

successful acclimatization plantlets were transferred to earthen pots containing

sterilized garden soil and garden manure and maintained in green house with

85% survival rate.

Similary, in C. sophera (Parveen and Shahzad 2010), rooting was best (93.6%)

obtained on half strength MS medium comprised of 1.0 µM IBA with the

production of maximum 5.7 ± 0.5 roots/shoot having 5.6 ± 0.5 cm root length

after 6 weeks. Regenerated plantlets with well-developed root system were

isolated from the rooting medium and hardened off inside the growth room. After

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acclimatization, plantlets were successfully transferred to greenhouse conditions

with 90% survival rate.

In C. tora (Quraishi et al. 2011), healthy and elongated roots with secondary

branches were recorded in 100% shoots on half strength MS medium

supplemented with 2.5 or 4.9 µM IBA. Plantlets with well-developed root system

were acclimatized and transferred to green house with 70% survival rate.

2.4 Different strategies of micropropagation in other plant species

2.4.1 Direct regeneration

In vitro regeneration without an intervening callus phase resulted in the direct

induction of shoots from various explants and produced genetically identical

plants (Hu and Wang 1983). Direct regeneration may occur either through pre-

existing meristems (axillary/shoot tip meristems) or from the well differentiated

tissues (leaf, stem, cotyledon, petiole, root etc.) lacking the meristem, in that

case it is referred as adventitious regeneration.

2.4.1.1 Apical meristem/axillary bud proliferation

Shoots of all angiosperms and gymnosperms grow by virtue of their apical

meristem. The apical meristem is usually a dome of tissue located at the

extreme tip of a shoot and measures approximately 0.1 mm in diameter and

approximately 0.25-0.30 mm in length. The apical meristem together with one to

three leaves primordial measuring 0.1-0.5 mm constitutes the shoot apex. The

first report of apical meristem culture was obtained in 1946 by Ball (Ball 1946).

He successfully raised transplantable whole plants of Lupinus and Tropaeoleum

by culturing their shoot tips with a couple of leaf primordial. However, Morel

together with Martin (Morel and Martin 1952) further refined this technique and

for the first time recovered virus free Dahlia plants by culturing shoot tips in vitro.

This technique of apical meristem culture since then widely been used with a

variety of plant species and has become the most efficient technique for

obtaining completely virus free plants (Belkengren and Miller 1962; Mullin et al.

1974 and Boxus et al. 1977). G Morel was the pioneer in applying shoot tip

culture for micropropagation of orchid Cymbidium (Morel 1965).

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Shoot proliferation from isolated apical or axillary bud under the influence of

different PGRs is the most frequently used micropropagation method for

commercial mass production of plants as it ensures maximum genetic uniformity

of the resulting plants. It involved the stimulation of axillary buds, which are

usually present in the axil of leaves, to develop into a shoot. In nature axillary

buds remain dormant for various periods depending upon the growth pattern of

the plant. In some plants, removal of terminal bud is necessary to break the

apical dominance and stimulate the axillary bud to grow into a shoot.

2.4.1.1.1 Effect of cytokinins and auxins on shoot multiplication

Wickson and Thimann (1958) showed that the growth of axillary buds, which

remain dormant in the presence of terminal buds, can be initiated by the

exogenous application of cytokinins. Since then a large number of pants have

been successfully micropropagated using cytokinins either singly or in

combination with an auxin. Cytokinins were first discovered by F Skoog, C Miller

and co-workers during the 1950’s as a factor that encourage cell division. The

first cytokinin discovered was an adenine derivative (aminopurine) named kinetin

(6-furfurylaminopurine) which was isolated as a DNA degradation product. The

first common natural cytokinin recognized was purified from immature maize

kernel and named zeatin. Cytokinins are present in all plant tissues. They are

abundant in root tips, shoot apex and immature seeds. Their endogenous

concentration is in the low nM range (Schmüling 2004). Naturally occurring

cytokinins are adenine derivatives with a side chain at the N6-position. An

example of synthetic cytokinin is benzyladenine or 6-benzylaminopurine (BA); it

is more stable and often used in plant tissue culture. In addition there are the

structurally unrelated phenylurea type cytokinins (eg. diphenyl urea, thidiazuron)

a class of synthetic cytokinins. In vitro the ratio of cytokinin to auxin regulates the

differentiation of cultured plant tissues to either shoots or roots. A high cytokinin

to auxin ratio promotes shoot formation, a low ratio, root formation.

Due to their stimulatory effect on plant regeneration cytokinins are extensively

used in plant tissue culture. Micropropagation of various plant species, including

many medicinal plants, has been accomplished through rapid proliferation of

shoot tips and axillary buds in culture during the last few years. Shoot tip

explants have been successfully employed in the regeneration of Catharanthus

30

roseus (Bajaj et al. 1988), Picrorhiza kurroa (Upadhyay et al. 1989),

Clerodendrum colebrookianum (Mao et al. 1995), Trichopus zeylanicus

(Krishnan et al. 1995), Withania somnifera (Kulkarni et al. 2000), Piper longum

(Soniya and Das 2002), Decalepis hameltonii (Giridhar et al. 2005), Eclipta alba

(Husain and Anis 2006a), Saussurea lappa (Johnson et al. 1997), Swertia

chirata (Balaraju et al. 2009), Aloe vera (Gantait et al. 2010) and Clitoria ternatea

(Anand et al. 2011). A large number of medicinal plants have been successfully

regenerated using axillary meristem either through nodal segment culture or

cotyledonary node (derived from aseptic seedlings) culture; a few of these plants

are Acacia nilotica (Dewan et al. 1992), Tylophora indica (Sharma and Chandel

1992), Oscimum basilicum (Sahoo et al. 1997), Dalbergia sissoo (Pradhan et al.

1998a), Psoralea corylifolia (Saxena et al. 1998), Gymnema sylvestre

(Komalavalli and Rao 2000), Acacia sinuata (Vengadesan et al. 2002),

Pterocarpus marsupium (Chand and Singh 2004a; Anis et al. 2005), Mucuna

pruriens (Faisal et al. 2006a), Glycyrrhiza glabra (Vadodaria et al. 2007), Acacia

senegal (Khalafalla and Daffalla 2008) and Veronica anagallis-aquatica

(Shahzad et al. 2011).

Numerous factors are reported to influence the success of in vitro propagation of

different medicinal plants and therefore, it is unwise to define any particular

reason for the general micropropagation of medicinal plants. The factors that

influence the micropropagation of medicinal and aromatic plants have been

reviewed by Murashige (1977), Hussey (1980; 1983), Hu and Wang (1983),

Bhagyalakshmi and Singh (1988), Short and Roberts (1991), Rout et al. (2000),

Chaturvedi et al. (2007), Khan et al. (2009), Sharma et al. (2010) and Krishnan

et al. (2011). Amongst all the cytokinins, BA, Kn and 2iP are most frequently

used for the micropropagation of different plant species, while, zeatin is rarely

used. On the basis of earlier studies it has been well documented that BA is the

most potent cytokinin for multiple shoot regeneration and had been successfully

used by several workers for the development of efficient micropropagation

protocols for various medicinal plants. Generally, a critical level of the hormone

is required for the induction of multiple shoots and for this a wide range of

concentrations has to be tested to select the best or optimal concentration of the

hormone.

31

Between two cytokinins (BA and Kn) tested by Agrawal and Sardar (2003) for in

vitro regeneration of Cassia angustifolia from various explants, 1.0 µM BA was

found to be optimal for eliciting best morphogenic response from seedling

derived CN explants. An average of 2.4 shoots/CN explant, 1.5 shoots/NS

explant and 1.08 shoots/ST explants were obtained at 1.0 µM of BA. The

superiority of BA over Kn has also been reported in other medicinal genera of

family Fabaceae (Leguminosae) viz: Bauhinia variegata (Mathur and

Mukunthakumar 1992), Psoralea corylifolia (Jeyakumar and Jayabalan 2002),

Sesbania rostrata (Jha et al. 2004), Acacia senegal (Khalafalla and Daffalla

2008), Acacia catechu (Jain et al. 2009a) and Clitoria ternatea (Pandeya et al.

2010).

In the micropropagation of an endangered medicinal plant Curculigo orchioids

(Wala and Jasrai 2003), single BA treatment was found to be suitable for the

induction of multiple shoots from meristem tip culture on MS medium containing

BA (2.21 µM). Faisal et al. (2006b) reported the development of 23.3 shoots/NS

at 5.0 µM of BA in Mucuna pruriens, Raghu et al. (2006a) obtained 6.3

shoots/NS at 8.87 µM of BA in Tinospora cordifolia, while Shahzad et al. (2011)

reported the production of a very high number of shoots (43.7 shoots/NS) at very

low concentration (0.5 µM) of BA in an amphibious medicinal plant Veronica

anagallis-aquatica. The edge of BA over other cytokinins is being well

documented in various other medicinal plants including Gymnema sylvestre

(Komalavalli and Rao 2000), Cardiospermum halicacabum (Babber et al. 2001),

Holostemma ada-kodien (Martin 2002), Ceropegia spp. (Beena et al. 2003 and

Nikam et al. 2008), Spilanthes acmella (Deka and Kalita 2005), Eclipta alba

(Dhaka and Kothari 2005), Bupleurum kaoi (Chen et al. 2006), Penthorum

chinense (Cao et al. 2007), Marsdenia brunoniana (Ugraiah et al. 2010) and

Ricinus communis (Alam et al. 2010).

The percentage bud break and multiple shoot induction declined with the

increase in BA concentration beyond the optimal level (2.0 mg/l) in Vitex

negundo (Sahoo and Chand 1998a) and suppressed the sprouting of dormant

axillary buds in the nodal explants. Reduction in the number of regenerated

shoots from apical or axillary meristems at a concentration higher than the

optimal level has also been reported in many leguminous plants (Gulati and

32

Jaiwal 1994; Kathiravan and Ignacimithu 1999; Sinha et al. 2000 and Anis et al.

2005) and several other medicinal plants like Kaempferia galanga (Vincent et al.

1992), Pisonia alba (Jagadishchandra et al. 1999), Cunila galioides (Fracaro and

Echeverrigaray 2001), Salvia guaranitica (Echeverrigaray et al. 2010) and

Veronica anagallis-aquatica (Shahzad et al. 2011).

Kumar (1992) and Nandwani and Ramawat (1993) reported that kinetin was the

best cytokinin for in vitro establishment and growth of leguminous trees such as

Bauhinia purpurea and Prosopis cinerarea respectively. Lal and Ahuja (1996)

reported a rapid proliferation rate in Picrorhiza kurroa using kinetin at 1.0-5.0

mg/l. Similarly, in the micropropagation of a medicinal plant Phyllanthus urinaria

(Catapan et al. 2002) a maximum of 20.3 shoots/NS explant were obtained on

MS medium supplemented with 5.0 µM Kn. Thangavel et al. (2011) also

obtained maximum number (19.7) of shoots per leaf explant on MS medium

fortified with 1.5 mg/l Kn with 80% regeneration percentage in the

micropropagation of a valuable medicinal herb Plectranthus barbatus. The effect

of 2iP on multiple shoot regeneration has been reported by Cellarova and

Hocariv (2004) in Digitalis purpurea and by Sujatha and Kumari (2008) in

Artemisia vulgaris.

One of the interesting observations was made by Vengadesan et al. (2002) in

the study of Acacia sinuata where multiple shoots were produced on MS medium

containing a combination of cytokinins (BAP and Kn). They observed that

maximum number of shoots was induced from CN explants on medium

containing 6.66 µM BA and 4.65 µM Kn. Similarly, Chaudhuri et al. (2007)

reported 18 shoots per NS explant in an endangered medicinal herb Swertia

chirata on MS medium containing BA (0.44 µM) + Kn (4.65 µM). In another

report by Balaraju et al. (2009) in the same plant the highest number of shoots

(42.16) per explant was also produced on MS medium containing 1.0 mg/l BA

and 0.1 mg/l Kn. BA + Kn combination has proved to be an ideal combination in

the micropropagation of Feronia limonia (Hossain et al. 1994) through cotyledon

explants excised from aseptic seedlings. However, Joshi and Dhawan (2007a)

reported maximum shoot multiplication in S. chirayita on medium containing 4.0

µM BA and 1.5 µM 2iP. Rajeswari and Paliwal (2006) reported the highest

frequency for shoot regeneration (82.5%), maximum number of shoots per CN

33

explant (6.9) and maximum shoot length (2.55 cm) in Albizia odoratissima on MS

medium containing 10.0 µM BA and 10.0 µM 2iP. Combination of two or more

cytokinins also favoured multiple shoot proliferation in Eucalyptus grandis

(Teixetra and Da Silva 1990), Fragaria indica (Bhatt and Dhar 2000), Eclipta alba

(Baskaran and Jaybalan 2005), Eucalyptus impensa (Bunn 2005), Ocimum

sanctum (Girija et al. 2006), Stevia rebaundiana (Ahmed et al. 2007), Amygdalus

communis (Akbas et al. 2009), Withania coagulans (Jain et al. 2009b), Cadaba

heterotricha (Abbas and Qaiser 2010) and Streblus asper (Gadidasu et al.

2011).

Generally cytokinin is required in optimal quantity for shoot proliferation in many

genotypes but inclusion of low concentration of auxins along with cytokinin

triggered the rate of shoot proliferation (Tsay et al. 1989; Roja et al. 1990 and

Shasany et al. 1998). Maximum percentage of multiple shoots (85.6%) was

observed by Rout (2005) in the medium supplemented with 8.9 µM BA and 1.34

µM NAA in a medicinal legume Clitoria ternatea. Addition of NAA (0.5 µM) along

with BA (5.0 µM) significantly enhanced the number of shoots (23.3) in another

medicinal plant Mucuna pruriense (Faisal et al. 2006b). Rapid proliferation of

shoots was achieved by culturing the in vitro shoots derived from the nodal

segments onto MS medium supplemented with 2.0 mg/l BA and 0.5 mg/l NAA in

the micropropagation of an important medicinal plant Gynura procumbens (Keng

et al. 2009). An average of 18.2 shoots was produced from each shoot explant.

The effective role of NAA in combination with BA for the induction of multiple

shoots has been reported in several other medicinal plants like Gomphrena

officinalis (Mercier et al. 1992), Gymnema sylvestre (Reddy et al. 1998),

Hemidesmus indicus (Sreekumar et al. 2000), Rauvolfia tetraphylla (Faisal and

Anis 2002), Ceropegia bulbosa (Britto et al. 2003), Justicia gendarussa

(Agastian et al. 2006), Bupleurum distichophyllum (Karuppusamy and Pullaiah

2007), Baliospermum montanum (George et al. 2008b), Boerhaavia diffusa

(Biswas et al. 2009), Spilanthes mauritiana (Sharma et al. 2009) and Stevia

rebaundiana (Sharma and Shahzad 2011). A rapid in vitro propagation of

Abelmoschus moschatus through axillary bud multiplication has been

established by Maheshwari and Kumar (2006) using MS basal medium

supplemented with different combinations of BA, NAA and IAA. Almost all

34

combinations responded but MS medium with 4.0 mg/l BA + 0.1 mg/l NAA was

the best suited for axillary bud proliferation inducing a mean of 15 shoots/node

which on further subculture generated more than 25 shoots/node. IAA also

favoured shoot induction but to a lesser extent as compared to NAA. Shoot

elongation was carried out on MS medium without PGRs. Ahmad and Anis

(2007) reported 25 shoots/NS explant in Vitex negundo at 1.0 µM of TDZ, but

the optimum shoot multiplication and elongation was achieved when TDZ

exposed cultures were subcultured on MS medium containing a combination of

BA (1.0 µM) and NAA (0.5 µM).

In contrast to the above mentioned studies some workers reported that the

combination of BA and IAA on MS medium favoured multiple shoot buds in

Adhatoda beddomei (Sudha and Seeni 1994), Alpinia galanga (Anand and

Hariharan 1997), Bupleurum fruticosum (Fratenale et al. 2002), Ocimum

gratissimum (Gopi et al. 2006), Acalypha wilkesiana (Sharma et al. 2007), Stevia

rebaundiana (Anbazhagan et al. 2010) and Clitoria ternatea (Anand et al. 2011).

High frequencies of multiple shoot regeneration were achieved from nodal

explants on MS medium fortified with 5.0 mg/l BA and 0.5 mg/l IAA in the

micropropagation of a medicinal climber Zehneria scabra (Anand and

Jeyachandran 2004). Eight to ten shoots per explant were obtained. An efficient

multiplication method from the shoot tips of Centaurium erythraea (Piactczaka et

al. 2005) using liquid MS medium supplemented with IAA (0.1 mg/l) and BA (1.0

mg/l) was developed from which a maximum of 60 shoots per explant were

produced.

Bohidar et al. (2008) obtained the highest number of shoots from nodal explant

of Ruta graveolens on MS medium supplemented with BA (1.0 mg/l) along with

IAA (0.25 mg/l). In the micropropagation of a woody medicinal plant (Aegle

marmelos), the highest shoot regeneration (86.6%) with an average shoot

number of 487.5 shoots per explant in seven week time was obtained on MS

medium supplemented with 6.6 µM BA and 1.14 µM IAA (Nayak et al. 2007).

The rate of shoot multiplication was greatly increased in a medium containing BA

(4.0-6.0 mg/l), IAA (1.0-1.5 mg/l) and adenine sulphate (AdS) (100 mg/l) in

Zingiber officinale (Palai et al. 1997). The addition of AdS at a concentration of

35

20.0 µM along with BA (4.0 µM) and IAA (0.5 µM) in MS medium improved the

rate of shoot multiplication in NS explant of Pterocarpus marsupium (Husain et

al. 2008). Not only vigour of shoots but proper leaf expansion and shoot

elongation was observed. The combination provided maximum response (85%)

with the highest number (8.6) of shoots with 4.8 cm shoot length after 6 weeks of

culture.

Beena et al. (2003) described in vitro regeneration protocol for Ceropegia

candelabrum through axillary bud multiplication using 8.87 µM BA in combination

with 2.46 µM IBA. The MS medium fortified with 1.0 mg/l BA and 0.5 mg/l IBA

provided maximum of 20 shoots/NS in a rare medicinal plant Rotula aquatica

(Martin 2003b). BA and IBA combination has also been described as the most

effective one in the micropropagation of Rheum emodi (Lal and Ahuja 1989),

Gardenia jasmenoides (George et al. 1993), Terminalia chebula (Shyamkumar

et al. 2003/2004) and Curcuma zedoria (Loc et al. 2005). Sen and Sharma

(1991) combined BA with 2,4-D for the multiplication of Withania somnifera.

Plantlet regeneration in Prosopis laevigata, a multipurpose leguminous tree, has

been achieved from cotyledonary nodes excised from in vitro grown seedlings

(Buendía-González et al. 2007). The explants were cultured on MS media

containing different concentrations of BA and 2,4-D and a mixture of organic

components. The highest number (3.37) of multiple shoots was observed in MS

medium containing 2,4-D (9.05 μM) + BA (6.62 μM).

Faster bud break coupled with an enhanced frequency of shoot development

(92%) and internode elongation was observed in Vitex negundo (Sahoo and

Chand 1998a). It was found to be dependent on the influence of Gibberelic acid

(GA3) when used at an optimal concentration (0.4 mg/l) along with BA (2.0 mg/l).

GA3 at 0.1-0.5 mg/l and AdS at 50-100 mg/l had a promising effect on shoot

proliferation and elongation (Rout et al. 2000). The promotive effect of GA3 in

combination with BA on shoot bud induction was also reported in

Chrysanthemum (Earle and Langhans 1974), Saussarea lappa (Arora and

Bhojwani 1989), Ocimum americanum (Pattnaik and Chand 1996), O. basilicum

(Sahoo et al. 1997), Tridax procumbens (Sahoo and Chand 1998b) and

Tylophora indica (Rani and Rana 2010). In contrast it has been reported to

suppress shoot bud differentiation at 0.1-0.3 mg/l in Plumbago indica (Nitsch and

36

Nitsch 1967), Begonia (Heide 1969), Nicotiana (Thorpe and Murashige 1970)

and Duboisia myoporoides (Kukreja and Mathur 1985). Thus, the role of GA3 in

shoot bud induction in plant species is controversial.

2.4.1.1.2 Effect of TDZ on shoot multiplication

Thidiazuron (TDZ) a substituted phenyl urea (N – phenyl-N-1,2,3 – thiadiazol – 5

–ylurea ) is one of the most active cytokinin-like substances that have been used

for high rate of axillary shoot proliferation in many plant species (Fiola et al.

1990; Malik and Saxena 1992a; Huettman and Preece 1993; Kim et al. 1997 and

Thomas 2003). TDZ was developed originally by AG Shering for use as a

defoliant for Gossypium hirsutum (Arndt et al. 1976). TDZ directly helps growth

due to its own biological activities in a manner similar to that of an N –

substituted cytokinin or it may induce the synthesis and accumulation of an

endogenous cytokinin (Capelle et al. 1983). In woody plant species, low levels of

TDZ induce the axillary shoot proliferation but higher levels may inhibit it. Higher

levels on the other hand encourage caulogenesis and somatic embryogenesis

(Huettman and Preece 1993; Lu 1993; Murthy et al. 1998; Shibli et al. 2001 and

Fengyun and Liying 2002). TDZ has been revealed to stimulate accumulation of

endogenous cytokinins (Murthy et al. 1995 and Hutchinson et al. 1996). In

addition to the cytokinin like activity, Hutchinson et al. (1996) observed that TDZ

promoted auxin accumulation. Other studies established that TDZ affected auxin

transport in hypocotyl tissues of Pelargonium x hortorum Bailey (Murch and

Saxena 2001) and encouraged the regeneration frequency by varying the levels

of abscisic acid (Li and Yang 1988), ethylene (Yip and Yang 1986) and proline

(Murch and Saxena 1997).

TDZ can be replaced for auxins or auxin-cytokinin required to stimulate somatic

embryogenesis. This is perhaps due to the association of TDZ in the modulation

of endogenous plant growth regulators especially auxins and cytokinins (Murthy

et al. 1995 and Chhabra et al. 2008). TDZ has been used in the range of 0.5-

10.0 µM to induce somatic embryogenesis from cotyledon explants of white ash

(Preece and Bates 1990 and Bates et al. 1992), eastern black walnut (Neuman

et al. 1993), Rubus (Fiola et al. 1990) and Vitis vinifera (Matsuta and Hirabayashi

1989). TDZ induced somatic embryogenesis has also been reported in several

other plant species like - geranium (Visser et al. 1992), Malus × domestica

37

(Daigny et al. 1996), Pelargonium x hortorum (Hutchinson et al. 1996), Prunus

avium × P. pseudocerasus (Pesce and Rugini 2004), Cicer arietenum (Kiran et

al. 2005) and Mangifera indica (Kidwai et al. 2009).

The mode of action of TDZ may be attributed to its ability to induce cytokinin

accumulation (Victor et al. 1999) and/or to enhance the accumulation and

translocation of auxin (Murch and Saxena 2001). The morphogenetic response

in which TDZ has been found to mimic cytokinin like activity was 20 times more

effective in dormancy breaking (Wang et al. 1986) and used successfully in plant

regeneration system of many plant species including several medicinal plants

viz: Hypericum perforatum (Murch et al. 2000), Bacopa monniera (Tiwari et al.

2001), Artemisia judaica (Liu et al. 2003), Arachis correntia (Mronginski et al.

2004), Curcuma longa (Prathanturarug et al. 2005), Hyoscyamus niger (Uranbey

2005), Sterculia urens (Hussain et al. 2007) and Andrographis neesiana

(Karuppusamy and Kalimuthu 2010), Similarly, TDZ has been proved to be

effective in regeneration of recalcitrant system such as grain legume (Malik and

Saxena 1992a). During last two decades it has been extensively used for the

high frequency shoot regeneration in various leguminous plants such as: Albizia

julibrissin (Sankhla et al. 1994), Cajanus cajan (Dolendro et al. 2003), Cicer

arietinum (Rizvi and Singh 2000 and Jayanand et al. 2003), Acacia sinuata

(Vengadesan et al. 2002), Robinia pseudoacacia (Hosseini-Nasr and Rashid

2003/4), Sesbania drummondii (Cheepala et al. 2004), Psoralea corylifolia

(Faisal and Anis 2006), Pterocarpus marsupium (Husain et al. 2007a) and

Leucaena leucocephala (Shaik et al. 2009).

The higher concentrations of TDZ has been proved to be inhibitory for multiple

shoot regeneration and decreased the shoot number in a number of plant

species like Phaseolus spp. (Malik and Saxena 1992a), Arachis hypogea

(Saxena et al. 1992) and Murraya koenigii (Bhuyan et al. 1997). Moreover, the

continuous or prolonged exposure of TDZ resulted in the distortion, stunting and

fasciation of shoots. Deleterious effects of TDZ have been well documented in

several plant species (Van Nieuwkerk et al. 1986; Preece et al. 1987; Yusnita et

al. 1990; Sankhla et al. 1994; Pradhan et al. 1998a; Ket et al. 2004; Khurana et

al. 2005 and Ahmad and Anis 2007). Addition of low concentration of an auxin or

a second cytokinin to the proliferation media containing TDZ significantly

38

enhanced shoot proliferation. Chalupa (1987) observed increased shoot

proliferation and elongation when BA + IBA or NAA were added to a TDZ

containing medium in Robinia pseudoacacia, Sorbus aucuparia and Tilia

cordata. Enhanced axillary shoot proliferation has also been reported in other

plant species when TDZ was added to a BA containing medium like: Acer x

fremanii (Kerns and Meyer 1987), Fraxinus americana (Navarrete et al. 1989),

Vitis rotundifolia (Sudarsono and Goldy 1991) and Hylocereus undatus

(Mohamed-Yasseen 2002).

2.4.1.1.3 Effect of subculture passages

A rapid rate of shoot multiplication and proliferation depends on the frequent sub

culturing of the regenerative shoot cultures. In case of prolonged cultures, the

nutrients in the medium are gradually exhausted and the same time relative

humidity in the vessels decreases leading to drying of the developing shoots for

lack of nutrients. Upadhyay et al. (1989) reported a propagation profile for

Picrorhiza kurroa and observed that the shoot multiplication rate gradually

improved as the number of sub culture passages increased. They proposed the

adaptation of the explants to the in vitro conditions, which was essentially

completed during the first few subcultures. Saxena et al. (1998) reported an

average of 3-5 fold multiplication in Psoralea corylifolia when axillary shoots

were allowed to continue in primary cultures for 8 weeks. Similarly, Shirin et al.

(2000) reported in vitro plantlet production in Kaempferia galanga on MS

medium containing 12.0 µM BA and 3.0 µM NAA and they observed 13 fold rate

of plantlet production every 4 weeks. Sub culturing of nodal segments harvested

from in vitro derived axenic shoots on the multiplication medium enable

continuous production of shoots in Centella asiatica (Tiwari et al. 2000).

Repeated sub culturing of shoot tips and single nodes of Cunila galoides at 4

weeks interval for eight months on the primary medium enable mass

multiplication of shoots without any evidence of decline (Fracaro and

Echeverrigaray 2001). In contrast Arya et al. (2003) demonstrated significant

shoot improvement in Leptadenia reticulata through repeated transfer of mother

explant to fresh medium. After three or four sub cultures, basal clump with shoot

bases was divided into three or four sub-clumps and multiplied on fresh medium.

They reported 15-20 shoots from each clump within 15 days.

39

2.4.1.2 Adventitious shoot regeneration

The induction of shoot buds from any site on the plant other than primary

meristem is termed as adventitious shoot regeneration. Buds are generally

induced directly from the explant without any intervening callus phase. Various

explants viz.: leaf, stem, internode, petiole, epicotyl, hypocotyl, cotyledon and

root are used as the source of adventitious shoot regeneration.

In Achras sapota (Purohit et al. 2004) direct shoot bud differentiation was

achieved through leaf segments on Schenk and Hildebrandt’s medium (SH

medium) supplemented with BA (8.88 µM) and TDZ (5.0 µM). Middle portion of

the leaves showed highest potential for shoot buds regeneration. Histological

observations also confirmed their de novo regeneration with clear vascular

connection with the mother tissues. The promotory effect of BA with IAA in

inducing direct shoot buds from leaf segments of Eryngium has been reported by

Arockiasamy et al. (2002). Similar results were also provided by Zhang et al.

(2005) in Perilla frutescens, where a maximum of 91.06% cotyledons induced

adventitious shoot buds on BA (4.44 µM) supplemented media while a

combination of BA (2.22 µM) and IAA (2.85 µM) was proved to be effective for

the regeneration of shoot buds from hypocotyl explant with 76.4% regeneration

in the same plant. 100% shoot regeneration was achieved in Verbena officinalis -

a medicinal plant (Turker et al. 2010) from stem internode explants, when BA

(13.32 µM) was combined with IAA (5.71 µM). While, BA along with AdS has

also found to be beneficial for shoot buds differentiation in Cajanus (Mishra

2002). A simple and efficient in vitro protocol was developed by Barik et al.

(2005) for high frequency shoot regeneration in Lathyrus sativus using epicotyl

segments. Highest shoot regeneration frequency (80%) was achieved on MS

medium containing BA (17.76 µM) and NAA (10.74 µM) with maximum shoot

regeneration (8.2 shoots/explant). Shoots were directly induced from the explant.

BA along with NAA has also been used by Burdyn et al. (2006) and Seetharam

et al. (2007) for direct shoot buds induction from leaf explants of Aloysia

polystachia and Vernonia cineria respectively.

However, in contrast to the above reports, Rao and Purohit (2006) suggested

that BA (4.44 µM) alone was effective in inducing direct shoot buds from

internode explants of Celastrus paniculatus. Incorporation of IAA and NAA did

40

not improve regeneration, rather promoted callusing. Similar results were also

obtained in different plant species (Espino et al. 2004; Purohit et al. 2004; Abdi

and Khosh-Khui 2007 and Sahai and Shahzad 2010). Bouhouche and Ksiksi

(2007) suggested the effective role of Kn (3.0 mg/l) in combination with IAA (0.5

mg/l) for the production of multiple shoots directly from hypocotyl explants of

Teucrium stocksianum, compared to other combination of PGRs. In Populus

deltoides, Yadav et al. (2009) also revealed that 0.25 mg/l Kn + 0.25 mg/l IAA

combination was most responsive for differentiation of direct adventitious shoot

buds from all along the explant surface in leaf, internode and root explants.

The effect of TDZ on adventitious shoot regeneration has been reported by

Debnath (2009) in a two-step procedure on excised leaves of lowbush blueberry.

TDZ induced cultures were transferred to medium containing 2.3-4.6 µM zeatin

and produced usable shoots after one additional subculture. Malik et al. (2010)

demonstrated the effective role of TDZ for direct shoot regeneration from intact

in vitro leaves (attached to shoots) of Arnebia euchroma. Shoot buds proliferated

to form multiple shoots on MS medium supplemented with Kn (5.0 µM). Direct

shoot regeneration was achieved when shoots were initially precultured for 40

days on higher concentrations of TDZ (20.0 µM) and then transferred to lower

concentration (5.0 µM). TDZ has also been used effectively to induce

adventitious shoot buds from leaf explant of many plant species (Huettman and

Preece 1993; Mithila et al. 2003; Gu and Zhang 2005 and Deore and Jhonson

2008).

2.4.2 Indirect organogenesis

The regeneration of plants via intermediate callus phase is termed as “indirecct

regeneration” or “indirect organogenesis”. The explants first dedifferentiate to

form an unorganised mass of cells called ‘callus’, the callus cells reorganize to

form ‘meristemoids’ which again redifferentiate to shoot buds, then finally

developed into shoots and regenerate plantlets. Various explants such as leaf,

stem, petiole, node, inter node, hypocotyl, root, cotyledon etc. can be used to

induce callus (Khurana et al. 2005). The induction of callus following

differentiation and organogenesis is accomplished by the differential use of

growth regulators and the control of conditions in the culture medium. With the

stimulus of endogenous growth substances or by addition of exogenous growth

41

regulators to the nutrient medium, cell division, cell growth and tissue

differentiation are induced (Tripathi and Tripathi 2003). Indirect organogenesis

often results in somaclonal variations, making the strategy less suitable for large

scale clonal propagation but at the same time the variants, thus, produced may

be utilized for the genetic improvement of species.

Callus mediated organogenesis has been reported in several medicinal plants

including Dioscorea deltoidea (Ravishankar and Grewal 1991), Datura innoxia

(Missaleva et al. 1993), Digitalis lantana (Pradel et al. 1997), Bacopa monnieri

(Shrivastava and Rajani 1999), Solanum nigrum (Shahzad et al. 1999), Ocimum

sanctum (Shahzad and Siddiqui 2000), Withania somnifera (Rani et al. 2003),

Tylophora indica (Faisal et al. 2005a), Ruta graveolens (Faisal et al. 2006c) and

Hypericum perforatum (Wojcik and Podstolski 2007). In Ceropegia candelabrum

(Beena and Martin 2003) and Decalepis hameltonii (Giridhar et al. 2004) callus

was induced from leaf and internodal explants and later the same callus

produced somatic embryos.

Saxena et al. (1997) regenerated plantlets of Psoralea corylifolia via callus

formation through mature leaves, stem, petioles and roots of young seedlings.

The callus differentiated into green nodular structure which developed into dark

green shoot buds in the medium comprised of MS + BA (2.5-3.0 mg/l) and NAA

(1.0 mg/l). Augmentation of AdS (5.0 mg/l) in the culture medium resulted in

quick growth of shoot buds within 4 weeks of culture. Pradhan et al. (1998b) and

Pattnaik et al. (2000) reported regeneration of plants from cell susupension

derived callus of Dalbergia latifolia and D. sissoo respectively. Vengadesan et al.

(2003a) and Shahzad et al. (2006) described efficient plant regeneration in a

medicinally valuable leguminous tree species Acacia sinuata from cotyledon

derived callus. Regeneration via callus phase has also been reported in other

members of Fabaceae family such as Sesbania bispinosa (Sinha and Mallick

1991), Lathyrus sativus (Roy et al. 1991; 1992), Dalbergia lanceolaria (Dwari

and Chand 1996), Acacia sinuata (Vengadesan et al. 2000), Leucaena

leucocephala (Saafi and Borthakur 2002 and Maity et al. 2005) and Clitoria

ternatea (Shahzad et al. 2007).

42

Generally auxins are considered as the best hormone for callus production. Hu

and Wang (1983) proved the superiority of 2,4-D over other auxins for induction

of callus and strongly antagonize any organized development. Shahzad et al.

(1999) suggested that 2,4-D at 2.0 mg/l was better than NAA at the same

concentration for the production of compact, nodular callus from the leaf explants

of Solanum nigrum. Similar findings were reported in Rauwolfia serpentina

(Perveen and Elahi 1987) and Plumbago rosea (Harikrishnan and Hariharan

1996). Similarly, 2,4-D was considered as the best auxin to induce organogenic

callus in case of Ceropegia bulbosa var lushii (Patil 1998) and Gymnema

sylvestris (Gopi and Vatsala 2006).

Addition of a cytokinin along with 2,4-D has found to be significant for callus

production, as in case of Acacia sinuata, Vengadesan et al. (2000) reported the

production of compact and nodular calli from hypocotyl explants on MS medium

comprised of 2,4-D and BA. Maximum number of shoot buds (12-15 per explant)

were differentiated on medium containing BA (13.3 µM) and IAA (3.42 µM) after

25-30 days of culture. The regenerated buds developed into shoots in less than

2 weeks after initiation. Faisal et al. (2006c) also suggested the production of

organogenic callus from stem explant of Ruta graveolens on MS medium

augmented with 2,4-D (10.0 µM) and BA (2.5 µM). Similarly, in Cerpegia juncea

(Nikam and Savant 2009), best organogenic callus was induced from nodal

segments on MS medium containing 2,4-D (1.0 µM) and BA (5.0 µM). However,

in Tylophora indica (Faisal et al. 2005a) the highest frequency (100%) of light

yellow organogenic callus was obtained from petiole explants by addition of TDZ

along with 2,4-D i.e. on medium comprised of MS + 2,4-D (10.0 µM) and TDZ

(2.5 µM). Inclusion of 2iP along with 2,4-D was also effective for callus

production in Pergularia daemia (Kiranmai et al. 2008). Baskaran and Jayabalan

(2009a) suggested the production of organogenic calli from leaf and petiole

explants in Melothria maderaspatana on MS medium containing 2,4-D (6.0 µM)

+ TDZ (0.5 µM) and 2,4-D (6.0 µM) + BA (1.0 µM) combinations respectively.

From these studies it has been proved that combination of 2,4-D and cytokinins

was efficient for the induction of callus and subsequent proliferation and

differentiation of shoots from such calli.

43

Inclusion of a cytokinin along with an auxin to induce callus has also been

reported in several other plant species (Gharyal et al. 1993; Sarasan et al. 1994;

Roy et al. 2000 and Hariharan et al. 2002). Rout et al. (1992) suggested the

callus production and subsequent plant regeneration in Cephalis ipecacuanha

from leaf explants cultured on MS medium containing Kn (4.5 mg/l) and NAA

(0.1 mg/l). Basu and Chand (1996) reported shoot buds differentiation from root

derived callus of Hyoscyamus muticus on MS medium comprised of BA (0.5

mg/l) and NAA (0.05 mg/l). Similarly, Irvani et al. (2010) achieved the best callus

production (100%) from root explants of Doreum ammoniacum on MS medium

supplemented with BA (2.0 mg/l) and NAA (1.0 mg/l).

In contrast to the above stated studies, Manjula et al. (2000) was of the opinion

that cytokinin alone was sufficient for the induction and subsequent growth of

callus in Tylophora indica and the inclusion of an auxin along with cytokinin

inhibited the growth of callus. Reddy et al. (2001) suggested that Kn alone

showed prominent growth of callus from leaf explants of Coleus forskohlii.

Addition of 2,4-D did not enhance the callus formation and the callus developed

on this medium subsequently turned brown within 4 weeks of culture. Shahzad

et al. (2006) also supported the above studies and produced callus from

cotyledon explants of Acacia sinuata on TDZ supplemented MS medium.

Efficient shoot buds production was observed when TDZ induced calli were

subcultured at different concentrations of BA.

Sood and Chauhan (2009) reported that addition of another auxin (IBA) along

with 2,4-D proved to be beneficial to induce callus cultures from different

explants such as leaf discs, nodal and root segments of Picrorhiza kurroa. There

are several other studies where inclusion of two auxins was proved to be optimal

for the induction of organogenic callus (Rani et al. 2003; Shahzad et al. 2009

and Ahmad et al. 2010).

2.4.3 Somatic embryogenesis

Somatic embryogenesis may be defined as a unique developmental pathway

that includes a number of characteristic events like: differentiation of cells,

activation of cell division and reprogramming of their physiology, metabolism and

give expression patterns. The somatic cells under suitable induction conditions

44

undergo restructuring through embryogenic pathway to develop embryogenic

cells. These cells then undergo a series of morphological and biochemical

changes that result in the formation of somatic embryo and generation of new

plants (Schmidt et al. 1997; Komamine et al. 2005 and Yang and Zhang 2010).

Somatic embryos resemble zygotic embryos and undergo almost the same

developmental stages (Dodeman et al. 1997). Regeneration via somatic

embryogenesis coupled with genetic engineering provides an opportunity of

producing a large number of elite or transgenic plants (Jin et al. 2005 and Li et

al. 2006). Sánchez et al. (2005) provided the first report on genetic

transformation of Quercus suber using somatic embryos through Agrobacterium

tumefaciens LBA4404/p35S GUS INT/pCAMBIA 1301 strain.

The first report of somatic embryogenesis in the history of plant tissue culture

was documented by Steward et al. (1958) and Reinert (1959) in carrot cell

suspension cultures. Since then somatic embryogenesis has been reported in a

wide range of plant species of dicot and monocot plants (Krishnaraj and Vasil

1995; Merkle et al. 1995; Quiroz-Figueroa et al. 2006 and Mathieu et al. 2006).

Somatic embryogenesis may involve the development of embryos either directly

from the explant without an intermediate callus phase or indirectly after a callus

phase and thus, referred as direct somatic embryogenesis (DSE) and indirect

somatic embryogenesis (ISE) respectively (Sharp et al. 1980). Chung et al.

(2007) documented plant regeneration through direct embryogenesis from leaf

explants of Dendrobium on MS medium containing 1.0 mg/l TDZ. Somatic

embryos were mostly found on cut ends near the leaf surface and occasionally

occurred near the leaf tips. DSE proved to be advantageous for the production of

true clones of the plants due to the minimal chances of changes in the genotype

(Peshke and Phillips 1992), however, reports of DSE are relatively rare (Gill and

Saxena 1992; Raghvan 1997; Chen et al. 1999; Chen and Chang 2006; Sudha

and Seeni 2006; You et al. 2007 and Varshney et al. 2009). Various explants

have been exploited for the induction of embryogenic calli but generally

immature, meristematic tissues proved to be the most suitable explant for

somatic embryogenesis. For instance immature zygotic embryos and cotyledons

have been used for the induction of somatic embryogenesis in majority of

legumes (Parrott et al. 1991; 1992; Neuman et al. 1993; Sagare et al. 1995; Rout

45

and Samantaray 1995; Ahmed et al. 1996 and Gairi and Rashid 2005). Choi et

al. (1999) reported the production of somatic embryos directly from the cotyledon

explants of Panax ginseng on growth regulator free medium.

Somatic embryogenesis results in the production of large number of plantlets

within short span of time. Regeneration of complete plantlets via somatic

embryogenesis has been successfully reported in a large number of medicinal

plant species (Ghosh and Sen 1991; Fuentes et al. 1993; Sehgal and Abbas

1994; Basu and Chand 1996; Kunitake and Mii 1997; Das et al. 1999; Jayanthi

and Mandal 2001; Tawfik and Noga 2002; Martin 2004a; Sudha and Seeni 2006

and Sahai et al. 2010a) including many leguminous plants of economic as well

as medicinal importance (Tetu et al. 1990; Durham and Parrott 1992; Arrilaga et

al. 1994; Dineshkumar et al. 1994; Garg et al. 1996; Rao and Lakshmisita 1996;

Mary and Jayabalan 1997; Luo and Jia 1998; Girija et al. 2000; Chand and

Singh 2001; Chand and Sahrawat 2002; Faisal et al. 2008 and Husain et al.

2010).

PGRs played an important role in the induction of embryogenic callus and then

subsequent conversion and development of somatic embryos to produce

complete plantlets. There are a number of species which showed

embryogenesis on medium supplemented with various cytokinins either singly or

in combinations. In an endangered medicinal plant Psoralea corylifolia

regeneration has been achieved via somatic embryogenesis through root

segments (Chand and Sahrawat 2002) on MS medium supplemented with NAA

(10.74 µM) and BA (2.2 µM) with highest frequency (95.2%) of embryogenic

calli. While Faisal et al. (2008) reported somatic embryogenesis and plant

regeneration in the same species from the nodal explants on MS medium

containing TDZ (16.0 µM). Further development of embryos to heart, torpedo

and cotyledonary stages was achieved on transferring to PGR free MS basal

medium within 2 weeks. Complete plantlets were regenerated from the somatic

embryos on half strength MS augmented with 1.0 µM GA3. Choi et al. (1999)

suggested that the requirement of GA3 for the germination of somatic embryos

was due to their dormant nature. The promotory effect of GA3 in somatic embryo

germination has also been reported in other plant species including Santalum

album (Rai and McComb 2002), Gossypium hirsutum (Kumaria et al. 2003) and

46

Panax japonicus (You et al. 2007). The high concentration of TDZ (7.5 µM) was

found to induce embryogenesis in Catharanthus roseus from mature zygotic

embryos, while the hypocotyl and cotyledon explants of the same species failed

to induce such response on TDZ supplemented media (Dhandapani et al. 2008).

Higher concentrations of TDZ have also been reported to induce somatic

embryogenesis in other plant species (Liu et al. 2003 and Mithila et al. 2003).

Contrary to these reports Gairi and Rashid (2005) observed high frequency (up

to 100%) direct somatic embryos regeneration at very low concentration of TDZ

(0.5 µM) from immature cotyledons of a highly medicinal trees species

Azadirachta indica. However, an increase in the concentration of TDZ to 1.0 µM,

further improved the regeneration potential.

Similarly, BA has also been reported to induce embryogenesis through various

explants in a number of other plants like Carthamus tinctorius (Mandal and

Gupta 2003) and Quassia amara (Martin and Madassery 2005). Inclusion of an

auxin along with BA found to enhance the rate of embryogenesis in Leptadenia

reticulata (Martin 2004a), wherein, somatic embryos at the highest frequency

was induced from ST and NS explants on MS medium supplemented with BA

(8.87 µM) and IBA (2.46 µM). While, differentiation of direct somatic embryos

from the nodal explant of a medicinal plant Hygrophila spinosa (Varshney et al.

2009) was observed on medium containing BA (1.0 µM) and NAA (0.5 µM).

Similarly, various cytokinins along with auxin found to facilitate induction of

embryogenic callus in a number of plant species (Samantaray et al. 1997;

Hussein and Batra 1998; Venkatachalam et al. 1999a; Nikam et al. 2003 and

Emek and Erdag 2007).

In general, auxins, particularly 2,4-D are considered as the most effective growth

regulators for the induction of embryogenic callus. 2,4-D has been proved to play

signalling role in induction of somatic embryos in many plant studies (Nomura

and Komamine 1995). The efficacy of 2,4-D in the induction of somatic embryos

and subsequent transferring of these embryos for further conversion and

development into plantlets on media comprised of different PGRs has been

observed in many reports (Choi et al. 1997; 2002; Martin 2003a; Chithra et al.

2005; Hu et al. 2008; Simões et al. 2010 and Naik and Murthy 2010). While,

there are reports available, in different species of plants where both the stages

47

i.e. induction and further development were achieved on the same medium

(Inamdar et al. 1990; Anbazhagan and Ganapathi 1999; PremAnand et al. 2000;

Magioli et al. 2001 and Park et al. 2005). But there are several reports available

in the literature, where 2,4-D was failed or proved to be insignificant for the

induction of embryogenesis ( Zhou et al. 1992; Chen et al. 1999; Kuo et al. 2005;

Sudha and Seeni 2006 and Varshney et al. 2009). However, a rapid and reliable

protocol for high frequency regeneration via somatic embryogenesis has been

developed in Mucuna pruriens, an important medicinal legume by Vibha et al.

(2009). They reported that embryogenic callus was induced from cotyledon

explants excised from axenic seedlings on MS medium containing 6.7 µM 2,4-D,

and a maximum of 60.5 cotyledonary staged embryos were obtained after 10

weeks of transfer on medium supplemented with 2.3 µM Kn and 5.4 µM NAA

along with 13.6 µM AdS. Mature embryos converted into complete plantlets on

half strength MS basal medium and exhibited 90% survival in field conditions.

Amoo and Ayisire (2005) reported successful callus production from the cut ends

of the cotyledon explant on 2,4-D supplemented medium. Calli turned friable and

nodular with small protuberances when transferred on media containing Kn

along with 2,4-D, while, further development of embryos was observed on auxin

free suspension culture medium. Similarly, augmentation of Kn along with 2,4-D

for efficient somatic embryogenesis has been studied in many plant species

(Toth and Lacy 1992; Hunault and Du Manoir 1992; Xie and Hong 2001a;

Jayanthi and Mandal 2001 and Naik and Murthy 2010). While, Husain et al.

(2010) achieved somatic embryogenesis in Pterocarpus marsupium from

hypocotyl explants cultured on MS medium augmented with 2,4-D (5.0 µM) and

BA (1.0 µM).

Maturation of somatic embryo and germination to produce complete plantlets is

the most crucial step of somatic embryogenesis. In some plant species MS basal

medium without any PGR was found to be sufficient for somatic embryo

germination and conversion into plantlets (Murthy and Saxena 1994 and Kim et

al. 2007). However, in many plant species reduction in MS salt composition (half

strength MS) proved to be beneficial for maturation of somatic embryos (Borad

et al. 2001; Yan et al. 2010a; Sahai et al. 2010a and 2010b). Augmentation of

BA to the germination medium also facilitates embryos maturation and

48

conversion as in case of Arachis hypogea (Venkatachalam et al. 1999a) and

Coriandrum sativum (Stephan and Jayabalan 2001). Addition of certain additives

like abscisic acid (ABA) along with BA also influenced the maturation of somatic

embryos in Vigna radiata (Girija et al. 2000). Mauri and Manzanera (2004) also

evaluated the effect of ABA and stratification during the maturation and

germination of holm oak (Quercus ilex). Stratification also promoted somatic

embryo germination in many other plant species like Rosa (Marchant et al. 1996)

and Quercus suber (Manzanera et al. 1993 and González-Benito et al. 2002). In

Medicago truncatula, BA along with NAA proved to be beneficial for somatic

embryo maturation and germination (Nolan et al. 1989). Nevertheless, García-

Martín et al. (2001) reported the effect of sucrose concentration, chilling

treatment and incubation condition on germination and conversion of somatic

embryos of Quercus suber.

2.5 Rooting in microshoots

Induction of roots in the regenerated microshoots is essential for the

development of complete plantlets and to make any regeneration protocol a

success. Various strategies have been applied by different workers to induce

healthy root system either under controlled conditions (in vitro) or in the external

environment (ex vitro) through the application of different auxins singly, in

combination or with different additives. A brief survey of literature showing

rooting in microshoots is described in the following heads:

2.5.1 In vitro rooting

Isolation of elongated shoots from the cultures and transferring to rooting media

comprised of different auxins facilitated in vitro root induction. However, the

microshoots of various medicinal plants have been successfully rooted on PGR

free MS basal medium (Cristina et al. 1990; Saxena et al. 1998 and Faisal and

Anis 2003). Reducing the strength of MS salts to half or one fourth or three

fourth also helped in the induction of roots. Half strength MS medium devoid of

any hormone induced rooting in Potentilla potaninii (He et al. 2006). Borthakur et

al. (2000) also reported that half strength MS medium was suitable for the

multiplication and growth of shoots with simultaneous rooting in Eclipta alba and

Eupatorium adenophorum. Half strength MS medium was found to be superior to

49

full strength MS medium for root induction and development in Mucuna pruriens

(Faisal et al. 2006a). Incorporation of IBA (2.0 µM) to the rooting medium

facilitated better rhizogenesis and maximum rooting percentage (92%) was

observed with fairly good length (5.5 cm) and number (7.8) of roots/shoot.

Presence of IBA in the medium found to induce rooting in many medicinal plants

including Sesbania acculeata (Bensal and Pandey 1993), Cajanus cajan

(Sivaprakash et al. 1994), Swaisona formosa (Jusaitis 1997), Gymnema

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

2000), Cunila galoides (Fracaro and Echeverrigaray 2001), Holostemma ada-

kodien (Martin 2002), Tylophora indica (Faisal and Anis 2003), Sesbania

drummondii (Cheepala et al. 2004), Eclipta alba (Baskaran and Jayabalan

2005), Psoralea corylifolia (Faisal and Anis 2006), Clitoria ternatea (Barik et al.

2007), Nolina recurvata (Bettaieb et al. 2008) and Vigna unguiculata (Aasim et

al. 2009). A combination of two auxins IBA (9.84 µM) and NAA (5.37 µM) in half

strength MS induced rooting in microshoots of Baliospermum montanum

(Johnson and Manickam 2003). While, Singh et al. (2003) suggested that full

strength MS basal medium was sufficient for root development in B. axillare.

In Citrus species (Thirumalai and Thumburaj 1996) rooting of regenerated

shoots was achieved on three fourth strength MS with NAA (3.0 mg/l). NAA was

found to be more effective than IBA when used singly for rooting in Scoparia

dulcis (Rashid et al. 2009). Efficient root induction was achieved on half strength

MS containing 0.5 mg/l NAA. In an earlier report, half strength MS fortified with

0.5 mg/l NAA induced a mean of 5.2 roots/shoot and the roots were well

branched with hairs in Rotula aquatica (Martin 2003b). Similar results were also

observed in other plants like Centella asiatica (Amin et al. 2003) and Tectona

grandis (Shirin et al. 2005). Peeters et al. (1991) concluded that NAA was taken

up six times faster than IAA and Van der Krieken et al. (1993) suggested that

IBA was taken up four times faster than IAA. Consequently the efficacy of NAA

may be due to its faster uptake (Martin 2003b). Higher rooting percentage (90%)

was obtained in Liquidamber styraciflua shoots cultivated in half strength WPM

with 0.5 mg/l NAA (Durkovic and Lux 2010). The efficacy of NAA at lower

concentration in rooting has also been reported in other medicinal plants like

Lippia alba (Gupta et al. 2001), Verbascum thapsus (Turker et al. 2001),

50

Safflower (Mandal and Gupta 2001), Santolina canescens (Casado et al. 2002)

and Ruta graveolens (Bohidar et al. 2008).

Swamy et al. (1992) observed the induction of roots in Dalbergia latifolia on IAA

supplemented medium. Similarly, Peternal et al. (2009) observed 60% rooting in

Populus tremula in half strength MS supplemented with IAA (1.0 µM) without

activated charcoal (AC). However, the effect of AC on root induction has been

reported in different plant species such as Rollina mucosa (Figueiredo et al.

2001), Curcuma zedory (Loc et al. 2005) and Swertia chirayitia (Joshi and

Dhawan 2007a and 2007b). But, the results of Balaraju et al. (2009) indicated

that no AC is required for rooting in S. chirata and the most effective rooting

(83%) was achieved on media comprised of MS + NAA (0.1 mg/l) with maximum

22.48 roots/shoot within 40 days.

To improve rooting percentage in Albizia odoratissima (Rajeswari and Paliwal

2006), a two-step method was adopted. Microshoots were treated with 25 µM

IBA for 24 hrs and then transferred to PGR free MS medium. Such shoot

exhibited highest 85% rooting and produced maximum number (6.15) of roots.

Similar results were also obtained in an earlier study by Sinha et al. (2000) in

Albizia chinensis, where microshoots were treated with 2.0 mg/l IBA in MS

medium and then subsequently subcultured in IBA free medium for the

development of efficient rooting. Two step rooting procedure was also found to

be effective in Quercus floribunda (Purohit et al. 2002). In another leguminous

tree species Pterocarpus marsupium (Anis et al. 2005), a pulse treatment with

an auxin IBA (200 µM) together with a phenolic acid for 5 days and subsequently

transferring to lower concentration of IBA (0.5 µM) on half strength MS was

proved to be efficient for 40-50% rooting. Husain et al. (2008) further suggested

the incorporation of (Phloroglucinol) PG along with IBA to facilitate better

rhizogenesis in the same plant in a two-step rooting procedure. Two-step rooting

procedure has also been used in several other studies (Shekhawat et al. 1993;

Choi et al. 2001; Romano et al. 2002; Husain and Anis 2004 and Husain et al.

2007a).

51

2.5.2 Ex vitro rooting

Ex vitro rooting provides is an alternative approach to induce rooting in

microshoots, it is economical, saves time and requires less labour, chemicals

and equipments. Ex vitro rooted plantlets did not require any additional step for

hardening and acclimatization prior to transplanting to the green house or field

conditions (Pruski et al. 2000). Like in vitro rooting, ex vitro rooting is also

affected by the chemical nature and concentration of auxins and the explant

source (Yan et al. 2010b) and it has been successfully applied in a variety of

plant species (Stapfer et al. 1985; Economou and Spanoudaki 1985; Zhang and

Davis Jr 1986; Shibli and Smith 1996; Kim et al. 1997 and Liu and Li 2001). For

the induction of ex vitro rooting different workers have emphasized the careful

selection of planting substrate and rooting treatment. The cut ends of the

microshoots were first dipped in different concentrations of rooting media

comprised of different auxins (IAA, IBA and NAA) followed by subsequent

transferring to sterile planting substrate. Successful ex vitro rooting has been

achieved in Siratia grosvenorii (Yan et al. 2010a) by NAA treatment. NAA has

also been reported to induce ex vitro rooting in Rotula aquatica (Martin 2003b).

But there are certain reports which suggested that IBA is more effective for ex

vitro root induction (Bhatia et al. 2002; Siddique and Anis 2006 and Ahmad and

Anis 2007). Contrary to all the above reports Feyissa et al. (2007) observed that

IAA is more effective for the induction of ex vitro roots in Hagenia abyssinica.

Martin et al. (2006) obtained 90% rooting in Celastrus paniculatus with 2-3 roots

of 2-4 cm in length through pulse treatment in a solution of 100 mg/l each of IBA

and NOA for 2 h and then for 3 min in 10 mg/l chlogengenic acid. Further they

reduced the cost and time involved in rooting by directly planting the in vitro

grown shoot tips of Celastrus paniculatus in polythene bags filled with river sand

and coir pith compost (1:1) and kept in humid chamber in green house. Within 9

days a maximum of 98-99% shoots were successfully rooted, hence reduced the

number of separate steps and the time for root induction and hardening.

2.6 Synthetic seeds

The encapsulation of in vitro derived propagules in a nutrient gel matrix leads to

the production of synthetic seeds or artificial seeds. Synthetic seeds also known

52

as ‘syn seeds’ may be defined as the artificially encapsulated somatic embryos,

shoot buds, cell aggregates or any other tissue that can be used for sowing as a

seed and possess the ability to convert into a plant under in vitro or ex vitro

conditions, and that retain this potential also after storage (Capuano et al. 1998

and Ara et al. 2000). Encapsulation provides protection and facilitates

conversion of in vitro derived propagules, therefore, encapsulation matrix must

contain nutrients, growth regulators and other components necessary for

germination and conversion of synthetic seeds (Ara et al. 2000). A number of

substances like potassium alginate, sodium alginate, carrageenan, agar, gelrite,

sodium pectate etc. have been used as encapsulation matrix but sodium alginate

(Na2-alginate) obtained from brown algae is the most suitable and is being

widely used at present (Redenbaugh 1993).

Encapsulation technology provides an effective means for in vitro germplasm

conservation, easy handling, exchange of genetic material between laboratories,

short or long term storage and direct transfer of in vitro material to ex vitro

conditions (Standardi and Picconi 1998; Ara et al. 2000; Chand and Singh

2004b; Rai et al. 2009 and Germaná et al. 2011). During the last 25 years

intensive researches have been made in the field of synthetic seed technology.

This technology provides a new dimension for future plant production and can be

applied to reduce the need for transplanting and subculturing during off season

periods. During cold storage, encapsulated nodal segments requires no transfer

to fresh medium, thus reduces the cost of maintaining germplasm cultures (West

et al. 2006). The first report of synseed production was made by Kitto and Janick

(1982), describing the encapsulation of carrot somatic embryos followed by their

desiccation. Since then synthetic seeds have been widely utilized for the mass

multiplication and conservation of a large number of plant species including

many medicinal plants such as Ocimum species (Mandal et al. 2000), Adhatoda

vasica (Anand and Bansal 2002), Rauvolfia tetraphylla (Faisal et al. 2006d)

Phyllanthus amarus (Singh et al. 2006a), Withania somnifera (Singh et al.

2006b), Rauvolfia serpentina (Ray and Bhattacharya 2008) and Cannabis sativa

(Lata et al. 2009a).

Somatic embryos are largely favoured for the production of synthetic seeds as

these structures possess both radicle and plumule that are able to develop into a

53

root and shoot in one step, without any pre-treatment (Redenbaugh 1993).

Encapsulation of somatic embryos for the production of artificial seeds has been

reported in many plants including Arachis hypogea (Padmaja et al. 1995),

Camellia japonica (Janeiro et al. 1997), Carica papaya (Castillo et al. 1998),

Asparagus cooperi (Ghosh and Sen 1994a), Arnebia euchroma (Manjkhola et al.

2005), Rotula aquatica (Chithra et al. 2005) and Quercus suber (Pintos et al.

2008). Various types of synseeds have been prepared using somatic embryos

which have been either dried (Gray 1987 and Senaratna et al. 1995) or

maintained fully hydrated (Redenbaugh 1990; Ghosh and Sen 1994a; Padmaja

et al. 1995; Onay et al. 1996 and Ara et al. 1999), these may or may not be

encapsulated (Kitto and Janick 1985a; 1985b; Redenbaugh et al. 1987 and

Bapat and Rao 1992).

In many plant species the unipolar structures such as hairy roots (Uozumi et al.

1992 and Nakashimada et al. 1995), axillary shoot buds (Ahmad and Anis 2010

and Singh et al. 2010), apical shoot tips (Rai et al. 2008 and Singh et al. 2009)

and protocorm like bodies (Sarmah et al. 2010) have also been encapsulated to

produce synthetic seeds. Since, axillary buds and shoot tips do not have root

meristems they should be induced to regenerate roots before encapsulation.

Piccioni (1997) and Capuano et al. (1998) described the conversion of shoot

buds of apple clonal root stocks, encapsulated after an appropriate root induction

treatment with IBA (24.6 µM) for 3-6 days. However, Bapat and Rao (1990) and

Ganapathi et al. (1992) described the conversion of encapsulated shoots buds of

Mulberry and Banana into plantlets without any specific root induction

treatments. A number of woody plant species have been successfully

propagated from artificial seeds containing in vitro shoot tips or nodal segments,

like Cedrela odorata (Maruyama et al. 1997a; 1997b), Coffea arabica (Nassar

2003), Dalbergia sissoo (Chand and Singh 2004b), Hibiscus moschetus (West et

al. 2006), Olea europea (Micheli et al. 2007 and Ikhlaq et al. 2010) and

Corymbia torelliana x C. citriodora (Hung and Trueman 2012).

Potential advantages of synthetic seeds include their designation as ‘genetically

identical materials’ ease of handling and transportation, along with increased

efficiency of in vitro propagation in terms of space, time and labour and overall

cost (Nyende et al. 2003). Faisal and Anis (2007) reported the regeneration of

54

Tylophora indica from encapsulated nodal segments collected from two years

old plant. Ideal beads were produced using 3% sodium alginate and 100 mM

calcium chloride. Maximum of 91% beads were converted into plantlets on MS

medium containing 2.5 µM BA and 0.5 µM NAA. Similar combination of 3%

sodium alginate and 100 mM calcium chloride was also tried earlier by Faisal et

al. (2006d) for the encapsulation of NS of Rauvolfia tetraphylla to produce ideal

and uniform beads. Lower concentrations of Na2-alginate resulted in the

production of soft and fragile beads which were difficult to handle while the

higher concentrations produced harder beads which retarded the regeneration

frequency. Maximum conversion (75.3%) of beads into multiple shoots was

recorded on MS medium containing BA (10.0 µM) and NAA (0.5 µM). An

encapsulation matrix of 5% Na2-alginate with 50 mM CaCl2.2H2O with ion

exchange duration of 30 min was found to be suitable for the formation of ideal

beads in Cannabis sativa (Lata et al. 2009a). However, in Picrorhiza kurroa

(Mishra et al. 2011), in vitro grown microshoots (ST and NS) were encapsulated

in the alginate beads containing 3% Na2-alginate and 3% CaCl2.2H2O. After 3

months of storage at 25 ± 2ºC in moist conditions, the encapsulated explants

were capable of regrowth within 2 weeks of following cultures, the frequency of

regrowth was 89.33% after 4 weeks of culture on PGR free MS medium.

Synseed technology offers an effective tool for the germplasm conservation and

short term storage for important medicinal plant species. Therefore, storage of

propagules at appropriate temperature and duration is essential to maintain the

viability of germplasm during exchange of material between laboratories and

also for future germination of synseeds. The percent conversion of encapsulated

NS into complete plantlets was quite high in comparison to non-encapsulated

explants after 4 weeks of storage at 4ºC as reported in Tylophora indica (Faisal

and Anis 2007). This report was in consonance with several earlier reports

(Mandal et al. 2000; Chand and Singh 2004b; Faisal et al. 2006d; Singh et al.

2006a and 2006b) which also supported the fact that cold storage of

encapsulated explants was more beneficial than storing at room temperature. In

most of the studies, 4ºC temperature was found to be the best for synseeds

storage (Saiprasad and Polisetty 2003; Kavyashree et al. 2006; Singh et al.

2007; Pintos et al. 2008; Sharma et al. 2009; Ikhlaq et al. 2010 and Tabassum et

55

al. 2010). However, Hung and Trueman (2012) suggested that storage of

synthetic seeds of Corymbia torelliana x C. citridora at 25ºC was much more

effective than refrigeration at 4ºC. Direct transfer of synseeds to the external

environment or ex vitro sowing in the sterile potting media (compost/potting

mix/vermiperlite) helped in the elimination of in vitro culture passages but its

application is limited due to its high cost of sterilising potting media (Hung and

Trueman 2012). However, ex vitro conversion of synthetic seeds has been

reported in many plant species (Mandal et al. 2000; Pattnaik and Chand 2000;

Soneji et al. 2002 and Naik and Chand 2006). Earlier, Bapat and Rao (1990)

reported 60% conversion of encapsulated axillary buds of mulberry under non

sterile conditions in autoclaved alginate matrix MS medium without sucrose.

Similarly, Lata et al. (2009a) obtained 100% conversion of synseeds of Cannabis

sativa on 1:1 potting mix composed of fermilome and coco natural medium.

2.7 Acclimatization

The ultimate success of micropropagation protocols depend on the ability to

transfer plants from in vitro to ex vitro or external environment with high survival

rates (Saxena and Dhawan 1999). Direct transfer of in vitro raised plantlets to

the field conditions is not possible due to high mortality rate as the plantlets

developed within the culture vessels were under low level of light, aseptic

conditions on a nutrient medium containing ample sugar to allow for

heterotrophic growth and in an atmosphere with high level of humidity. These

culture conditions resulted in plantlets with abnormal morphology, anatomy and

physiology (Pospíšilová et al. 1992; Desjardins 1995; Hazarika 2003 and

Chandra et al. 2010). The concentration of sucrose and agar in the medium also

affected the subsequent acclimatization to ex vitro conditions (Synková 1997 and

Lavanya et al. 2009). Regenerated plantlets on transferring to field or green

house conditions faced various abiotic (altered temperature, light intensity and

humidity conditions) and biotic (soil microflora) stress conditions. Direct transfer

to sunlight also causes charring of leaves and wilting of the plants. Thus, a

period of acclimatization or more specifically a period of transitional development

is required in which both anatomical characters and physiological performances

overcome the influence of in vitro culture conditions for the successful

56

establishment and survival of the plantlets (Donnelly et al. 1986; Hiren et al.

2004; Lavanya et al. 2009 and Deb and Imchen 2010).

The acclimatization period may vary from 15 days to 1 month depending upon

the nature and hardness of the plant species (Ziv et al. 1987; Donnelly and

Tisdall 1993; Nowak and Shulaev 2003 and Hazarika 2003). The in vitro raised

plantlets have non-functional stomata, weak root system and poorly developed

cuticle which resulted in high mortality of regenerants upon transfer to ex vitro

conditions (Mathur et al. 2008). Stomata are generally large with changed shape

and structure having guard cells with thinner cell wall and contain more starch

and chloroplast (Martin et al. 1988). During acclimatization to ex vitro conditions

leaf thickness gradually increases, leaf mesophylls differentiated into palisade

and spongy parenchyma, stomatal density decreased and became elliptical from

circular shape. Development of cuticle, epicuticular waxes and effective stomatal

regulation of transpiration occur leading to stabilization of water potential of field

transferred plantlets (Pospíšilová et al. 1999 and Chandra et al. 2010). Several

growth retardants can be used in micropropagation to reduce damage due to

wilting without deleterious side effects. Use of paclobutrazol (0.5-4.0 mg/l) in the

rooting medium is reported to reduce stomatal apertures, increase epicuticular

wax, short stem and thick roots (Smith et al. 1990a; 1990b; 1991 and Chandra et

al. 2010). Abscisic acid (ABA) a naturally occurring plant hormone played an

important role in plant water balance and in the adaptation of plantlets to stress

environments including low temperature (Hetherington 2001 and Finkelstein and

Gibson 2002). Various stresses induce ABA synthesis and considered as a plant

stress hormone (Tuteja 2007). Aguilar et al. (2000) studied the role of ABA on

tolerance to abiotic stress in Tagetes erecta in controlling leaf water loss,

survival and growth of microshoots when transferred directly to the field.

Various strategies have been applied by different workers for the successful

acclimatization and establishment of regenerated plants in field conditions.

Bhuyan et al. (1997) reported the acclimatization of plantlets of Murraya koenigii

in vermicompost inside the plant growth chamber for 3 weeks and then

subsequently established the plantlets in soil with about 85% survival rate. The

rooted plantlets of Yucca aloifolia (Atta-Alla and van Staden 1997) were treated

with 0.2% Benlate (Active ingredient, Benzimidazole) for 10 min to reduce fungal

57

contamination. These plantlets were then hardened off under mist culture media

containing loam soil, Sphagnum peat and pine bark in 1:1:1 ratio. After 3 weeks

of acclimatization plantlets were transferred to greenhouse conditions.

Komalavalli and Rao (2000) reported 80-85% survival of plantlets of Gymnema

sylvestre in the soil. While, Anitha and Pullaiah (2002a) observed only 40% plant

survival in Sterculia foetida in the field conditions after adopting a series of

hardening and acclimatization procedure. Rooted plantlets of S. foetida were

placed in liquid quarter strength MS basal medium and then transferred to pots

containing sterilized sand, soil and manure mixture (1:1:1). Plantlets were

irrigated with half strength MS basal medium and acclimatized to room

temperature for 14 days and later shifted to green house and kept under shade

for 30 days, then finally planted in soil with 40% survival. Rooted plantlets of

Tinospora cordifolia (Raghu et al. 2006a) were acclimatized in thermocol cups

containing sand and soil (1:1) for 14 days. After that plantlets were transferred to

nursery for 2 months and then transplanted in the soil with 80% plant survival.

Direct transfer of plantlets from culture vessels to pots showed a high rate of

mortality in Pterocarpus santalinus (Prakash et al. 2006). Thus, plantlets were

first acclimatized in the plant growth chambers for 2 weeks with high humidity

and other incubation conditions. Plantlets were then transferred to pots

containing a mixture of soil and farmyard manure (4:1) and kept under shade

area of forest nursery for about 4 weeks and exhibited 70% survival rate. About

90% of the micropropagated plants of Mucuna pruriens (Faisal et al. 2006b)

survived following transfer from soilrite to natural soil and did not show any

detectable variation in respect to morphology or growth characteristics. Khatun

et al. (2010) gradually shifted the micropropagated plants of Citrullus lanatus

from culture room to field conditions. Firstly, the plantlets were kept in growth

room for two days then transferred from growth room to open room and kept

there for four days. Plantlets were regularly sprayed with water and covered with

polythene bags to maintain higher humidity. Finally, the plants were gradually

acclimatized to outdoor conditions with 80% survival rate and satisfactory

growth.

58

2.8 Physiological studies

The transferring of in vitro raised plantlets from low light intensity (1200-3000 lux)

and controlled temperature (25 ± 2ºC) to broad spectrum sunlight (4000-12000

lux) and high temperature (26-36ºC) of external environment often resulted in the

charring of leaves and wilting of the plantlets. It also might lead to transient

decrease in photosynthetic parameters. Therefore, it is necessary to hardened or

acclimatized the plants under greenhouse or shade conditions before being

exposed to field environment (Lavanya et al. 2009 and Chandra et al. 2010). In

many plant species, the leaves formed in vitro are unable to develop further

under ex vitro conditions and are replaced by newly formed leaves (Preece and

Sutter 1991 and Diettrich et al. 1992). However, if the ex vitro transplantation of

plantlets is successful, the increase in their growth can be enormous for

example, total dry mass of Nicotiana tabacum plants was several times higher

than that of plantlets grown in vitro, the transplanted plants were taller, had

higher dry mass of leaves, stem and roots and larger leaf area and leaf thickness

(Kadleček et al. 1998). After transplantation of tissue culture plants to ex vitro

conditions, most of the plants develop a functional photosynthetic apparatus and

the contents of photosynthetic pigments increased, although the increase in light

intensity is not linearly translated in an increase in photosynthesis (Kozai 1991;

Trillas et al. 1995; Rival et al. 1997; Synková 1997; Pospíšilová et al. 1998 and

Amâncio et al. 1999).

In Calathea louisae the in vitro formed leaves were unable to photosynthesize

during the first days after transplantation but in Spathiphyllum floribundum the in

vitro formed leaves were photosynthetically capable and normal source-sink

relations were observed. Nevertheless, in both plant species, considerable

photosynthetic activities were measured when new leaves were fully developed

(Van Huylenbroeck et al. 1998). Further, Van Huylenbroeck et al. (2000)

reported three times higher contents of chlorophyll and carotenoids in ex vitro

formed leaves in comparison to in vitro ones and they observed an inverse

relation between PPFD and the chlorophyll/carotenoids ratio at the end of

acclimatization. In Solanum tuberosum and Spathiphyllum floribundum net

photosynthetic rate (PN) decreased in the first week of transplantation and

increased thereafter (Baroja et al. 1995; Van Huylenbroeck and Debergh 1996

59

and Pospíšilová et al. 1999). Transfer of in vitro raised plants to ex vitro

conditions under direct sunlight might cause photoinhibition and chlorophyll

photobleaching. After transplantation, the 14CO2 uptake by persistent leaves of

Fragaria and Rubus idaeus was similar to that in plantlets grown in vitro or was

slightly increased, and a significantly increased 14CO2 uptake was found only in

newly formed leaves (Short et al. 1984 and Deng and Donnelly 1993). The

above mention result suggests that photoinhibition might be the cause of the

transient decrease in photosynthesis after transplantation (Chandra et al. 2010).

2.9 Different factors affecting in vitro regeneration

2.9.1 Nature of explant: source, type and age of the explant

Khalafallah and Daffala (2008) observed that the CN explants of a leguminous

tree species Acaica senegal derived from 7 days old axenic seedlings provided

better response as compared to the NS explants collected from 12 months old

greenhouse grown plants. CN explants gave the highest number and longest in

vitro regenerated shoots compared to those induced from NS on the same

regeneration medium. Thus, the study suggested that young and juvenile

explants (CN) responded better than the mature explants (NS) and it appeared

that multiplication rate is feasible by using defined PGRs and supplements when

combined with appropriate physiological state of the explant. The rate of shoot

regeneration in Sterculia urens (Sunnichan et al. 1998) is relatively quite low (6.0

shoots/explant) when the explants (NS) were collected from mature tree

compared to the (11.24 shoots/CN) explants derived from in vitro grown

seedlings (Devi et al. 2011). The source and the type of explants had a great

influence on the induction and multiplication of shoots in vitro in different plant

species including many woody legumes such as Dalbergia sissoo (Pradhan et al.

1998a), Albizia lebbeck (Mamun et al. 2004), Pterocarpus marsupium (Anis et al.

2005) and Albizia odoratissima (Rajeswari and Paliwal 2006).

The rate of regeneration and morphogenetic response varied to a great extent

according to the type of explants. The differences in culture requirements exist

among different parts of the same plant may be attributed to the various levels of

endogenous plant growth regulators of explants from different positions (Ghosh

and Sen 1994b; Yucesan et al. 2007 and Lisowska and Wysokinska 2000).

60

According to Thengane et al. (2001) leaf explant was proved to be more

significant for the regeneration of adventitious shoots in Nothapodytes foetida

followed by hypocotyl and cotyledons. Vengadesan et al. (2002) suggested that

the percentage response varied with the type of growth regulators used, its

concentration and the type of explants. Between CN and ST explants derived

from axenic seedlings, CN explants provided better morphogenic potential for

multiple shoot development in Acacia sinuata. Sivanesan and Jeong (2007)

reported more number of shoots from NS explants as compared to ST explants

in Pentanema indicum. However, maximum shoot regeneration in Carlina acaulis

(Trejgell et al. 2009) and Senecio macrophyllus (Trejgell et al. 2010) was

reported from seedling derived ST explants in comparison to the hypocotyl,

cotyledon and root explants. In Stevia rebaudiana also, ST explants were proved

to be the most effective explant for maximum shoot regeneration than NS and L

explants when cultured on MS medium supplemented with 1.0 mg/l BA and 0.5

mg/l IAA (Anbazhagan et al. 2010). However, Gonçalves et al. (2010) reported

maximum shoot proliferation from NS than apical ST explants in Tuberaria

major.

Age of the different explants collected from in vitro grown seedlings had

differential response on multiple shoot regeneration in different plant species and

it varied according to the type of growth regulators used, their concentrations

and type of the explants. CN explants collected from 20 days old axenic

seedlings of Psoralea corylifolia (Jeyakumar and Jayabalan 2002) produced

multiple shoots on MS medium supplemented with different PGRs. A significantly

greater number of shoots were produced from cotyledons of 14 days old

embryos in mulberry on MS medium containing TDZ and BA compared to 7 and

21 days old explants (Thomas 2003). In Pterocarpus marsupium also, among

various explants used for the induction of multiple shoots, CN explants excised

from 18 days old seedlings provided excellent response in terms of highest

number of shoots/explant as well as maximum shoot length (Anis et al. 2005 and

Husain et al. 2007a). However, Chand and Singh (2004a) excised CN explants

of P. marsupium from 20 days old seedlings for multiple shoot regeneration.

Alam et al. (2010) used CN explants derived from 5-7 days old aseptic seedlings

of Ricinus communis for multiple shoot regeneration. Karuppusamy and

61

Kalimuthu (2010) developed an efficient regeneration protocol for an endemic

medicinal plant Andrographis neesiana through NS derived from 30 days old

seedlings. Husain et al. (2008) used 18 days old nodal segments excised from

axenic seedling in Pterocarpus marsupium. Different explants like leaf segments,

nodal segment and root segments derived from 5 weeks old seedlings were

used for the induction of callus in Citrus jambhiri by Savita et al. (2010).

Similarly, Prakash and Gurumurthi (2010) induced callus in Eucalyptus

camaldulensis using mature zygotic embryos and cotyledons explants collected

from 10, 15, 25 and 30 days old aseptic seedlings. The frequency of callus

induction decreased with the increasing age of the explants and the highest

frequency was obtained in 10 day old explants (60%) followed by 15 day old

explants (43%).

2.9.2 Media composition

The rate of regeneration often depends not only on the selection of the most

suitable explants but also on the composition of the basal medium. The

nutritional requirement varies according to the cell, tissues and organ and also

with respect to particular plant species (Basu and Chand 1996). A variety of

media and salt concentrations have been used, but MS medium still remain the

most widely used formulation for the multiplication of different plant species. The

B5 and WPM media are also used most frequently for the micropropagation of

several plants. Several researchers have used the modification of these media

or other formulations like White’s medium (White 1963), RWM (Risser’s and

White’s Medium 1964), LS (Linsmaier and Skoog Medium 1965), NN (Nitsch and

Nitsch 1969), SH (Schenk and Hildebrandt’s Medium 1972), L2 (Philips and

Collins 1979), AE (Von Arnold and Eriksson 1981) and UM (Litvay et al. 1985)

for axillary, adventitious as well as somatic embryogenesis etc. various mineral

salt formulations have been used for in vitro culture, however, full strength

mineral salts are not always optimum and different formulations may work better

at different stages (Thorpe et al. 1991). These combinations are used either at

full strength or with modifications based on experimental results. Modification in

the MS such as MS salts reduced to one half, one third, one fourth, one fifth or

three fourth have been found effective in different reports (Badji et al. 1993;

Hung et al. 1994; Das et al. 1996a and Faisal et al. 2007).

62

B5 medium has been used by several workers in the micropropagation of

important plant species and proved to be effective in the regeneration of

Pterocarpus marsupium (Lakshmi Sita et al. 1992), Acacia nilotica (Dewan et al.

1992 and Samake et al. 2011), P. santalinus (Anuradha and Pullaiah 1999),

Gardenia jasmonoides (Chuenboonngarm et al. 2001) and Jatropha curcas

(Warakagoda and Subasinghe 2009). Joshi et al. (2003) concluded that for the

establishment and elongation of shoots in a leguminous tree species, Dalbergia

sissoo, B5 medium is better than MS medium, while, the maximum number of

shoots were obtained on MS medium. Similar results were also obtained by

Berger and Schaffner (1995) in another leguminous tree species Swartzia

madagascariensis. However, Abbas et al. (2010) proved that MS medium was

more effective than B5 medium in A. nilotica for the production of maximum

number of shoots. Bhatt and Dhar (2004) suggested that WPM provided better

response than B5, MS and half strength MS in Myrica esculanta. Moreover,

WPM has also been used in a number of plant species as the regeneration

medium such as Ixora coccinea (Lakshmanan et al. 1997), Acacia catechu (Das

et al. 1996b), Cinnamomum camphora (Nirmal Babu et al. 2003), Tinospora

cordifolia (Raghu et al. 2006a) and Salix tetrasperma (Khan et al. 2011). Wang

et al. (2005) were of the opinion that WPM and B5 had better effects for shoot

regeneration than MS medium in Camptotheca acuminata and relatively higher

shoot buds were produced. Douglas and McNamara (2000) obtained

adventitious shoot regeneration in Acacia mangium by using Juglans medium

(DKW). Baskaran and Jayabalan (2008 and 2009b) used L2 medium for the

micropropagation of Psoralea corylifolia.

Tiwari et al. (2004) tested three different media (MS, B5 and White’s) with or

without different PGRs for the in vitro propagation of Pterocarpus marsupium

using NS explants. Amongst all the three media tested, MS medium was proved

to be the best for highest shoot regeneration. Among three different media (MS,

B5 and SH) tested by Baskaran and Jayabalan (2005) for the micropropagation

of Eclipta alba, MS medium was found to support better shoot regeneration than

B5 and SH media. MS medium has also found more effective than other media in

several other medicinal plants also (Gao et al. 1999; Komalavalli and Rao 2000;

Wondyifraw and Surawit 2004; Wang et al. 2008 and Perveen et al. 2011).

63

2.9.3 Sources of carbohydrate and their concentration

In vitro culture of plant cells, tissue and organ generally requires the addition of a

carbon or energy source to the culture medium (George 1993 and Karhu 1997).

Sucrose has been used as the most common source of carbon in plant tissue

culture probably, because it is the most common carbohydrate in the phloem sap

of many plants (Lemos and Baker 1998 and Fuentes et al. 2000). However,

besides sucrose, some additional carbohydrate sources like glucose, fructose,

maltose and sorbitol etc. has also been used by different workers as an

alternative source (Lou and Sako 1995; Hisachi and Murai 1996; Mamiya and

Sakamoto 2000; Vijaya Chitra and Padmaja 2001; Mosaleeyanon et al. 2004;

Anwar et al. 2005 and Mohamed and Alsadon 2010).

In most of the cases, optimum concentration (3%) of sucrose than 2 and 4% was

used for shoot multiplication and proliferation as recommended by Murashige

and Skoog (1962). Baskaran and Jayabalan (2005) reported that among three

different carbohydrate sources (glucose, fructose and sucrose), 3% sucrose was

found to be better for shoot regeneration than fructose and glucose.

Augmentation of 3% sucrose in the culture medium has been found to provide

best response in cork oak (Romano et al. 1995) and Kaempferia (Shirin et al.

2000). While, sucrose and glucose induced the highest frequency of shoot

regeneration in Bixa orellana (De Paiva Neto et al. 2003). There are many

researchers who used the various concentrations of sucrose either higher or

lower for shoot proliferation. Effect of different concentrations of sucrose was

studied by Gürel and Gülşen (1998) on in vitro shoot production of Amygdalus

communis during three successive stages of development. During the initiation

and transplantation stage, 5 and 6% sucrose gave the best results with respect

to shoot production and growth. During the multiplication stage, the highest rate

of shoot production was achieved with 3 and 4% sucrose. Beck et al. (1998)

reported that an increase in sucrose concentration increased the shoot

production in Acacia mearnsii. While, in some cases of somatic embryogenesis,

lower and higher concentrations of sucrose has been tested for specific

purposes as reported by Xie and Hong (2001a). Karami et al. (2006) suggested

that higher concentrations (9 to 15%) of sucrose improved maturation of somatic

embryos, while regeneration of complete plantlets from these embryos was

64

achieved on half strength MS medium without any PGR containing 3% sucrose

only. Higher concentrations of sucrose have also influenced somatic

embryogenesis in other plant species (Tremblay and Tremblay 1991; Ricci et al.

2002 and Biahoua and Bonneau 1999).

Tyagi et al. (2007) recommended the use of LR grade sucrose and sugar cubes

in Curcuma longa cultures. Joshi et al. (2009) tried to develop a

micropropagation protocol for Wrightia tomentosa using different carbon

sources, gelling agents and type of culture vessels in an effort to reduce the cost

of micropropagation. Observations revealed that the best regeneration was

achieved on the medium containing sugar cubes (3%) as the carbon source. The

promontory effect of sugar cubes in shoot multiplication has been previously

reported in Leucaena leucocephala (Dhawan and Bhojwani 1984). Sridhar and

Naidu (2011) observed that the highest number of shoots in Solanum nigrum

were obtained on MS medium supplemented with 4% fructose, while, maximum

shoot length was achieved on medium containing 4% sucrose.

2.9.4 pH of the medium

The pH of the medium has a promotory effect on nutrient uptake as well as

enzymatic and hormonal activities in plants. Plant cells and tissues require an

optimum pH for growth and development (Hussey 1986; Gürel and Gülşen 1998

and Bhatia and Ashwath 2005). Changes in the pH of the medium influenced the

performance and development of explants (George et al. 2008a). The optimum

pH level regulates the cytoplasmic activity that affects cell division and growth of

shoots and it does not interrupt the function of the cell membrane and the

buffered pH of the cytoplasm (Brown et al. 1979). Lazzeri et al. (1987) concluded

that the pH value had no significant effect on the efficiency of shoot regeneration

in soybean. But in most of the studies, pH value 5.8 was found to be the best for

shoot morphogenesis (Gautam et al. 1993; Nair and Seeni 2003 and Perveen et

al. 2011). However, certain plants require acidic pH for maximum regeneration

(Naik et al. 2010).

Sanavy and Moeini (2003) reported that the pH level of 5.5 was the best for the

overall growth of the plants in potato meristem culture. Low and high levels of pH

than 5.5 were found to reduce the growth and rooting. The reduction was more

65

pronounced at low levels than high levels of pH. Similarly, Huda et al. (2009)

also reported that the highest rate of shoot regeneration was achieved in a

medium comprised of 0.5 mg/l IAA and 3.0 mg/l BA at pH 5.5 in tossa jute. A

range of pH level from 5.8 to 6.6 was found to be effective for shoot regeneration

of Camptotheca acuminata (Wang et al. 2005), while, the best response (90%)

and a high shoot number was found on the medium at pH 5.8. At pH 7.0 and

below 5.4 the regeneration was low; moreover on the medium with pH value

below 5.4, regenerated shoots show vitrification. In chickpea (Barna and Wakhlu

1993) pH 6.5 was proved to be the optimum for embryo maturation which was

adversely affected by the pH above 7.0 and below 4.0.

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