Chapter - 2 REVIEW 2.1. In vitro re ... regeneration via organogenesis from callus culture derived from

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  • Chapter - 2 REVIEW

    Huge volume of works have been completed world wide about the application

    of tissue culture technology in in vitro regeneration of many plant species which are

    difficult to regenerate by conventional methods and save them from endangerment and

    extinction. Some of the representative works are presented here to justify the present


    2.1. In vitro regeneration

    Tissue culture technology is employed in three occasions for plants such as i) to

    make reproduction in the plant species of lower propagation efficiency and threatened

    category ii) to protect the wild by getting benefits from the in vitro developed callus and

    iii) to get the clones of desired characters by making gene manipulation at callus level.

    In addition, with micropropagation, the multiplication rate is greatly increased and it

    also permits the production of pathogen-free material (Nehra and Kartha, 1994).

    In vitro regeneration attempts and standardization of basal medium for the

    propagation of plant species with respect to red listed species and economically

    important species are under practice world wide (Cuenca et al., 1999; Naomita and Rai,

    2000). Mascarenhas and Muralidharan (1989) reviewed the tissue culture studies carried

    out in India for the manipulation of important forest species. de Oliveira et al. (2003)

    successfully employed the tissue culture techniques for an economically important plant

    species, Tabernaemontana fuchsiaefolia to in vitro regeneration in Brazil. Earlierly,

    Begum et al. (2002) standardized the basal medium for in vitro regeneration of

    ethnobotanically important pan tropical herbal species, Ocimum basilicum and the

    plantlets produced by this method were found to have 75% survivability in fields.

    Rahman et al. (2004) successfully developed callogenesis and organogenesis in

    Curcuma longa (turmeric) and the results of the hardening experiments revealed that

    over 70% of transplanted plantlets of this species was survived in the filed. The species,

    Spilanthes mauritiana is a native of Eastern Africa has wide medicinal uses with less

    population size in its homeland, the Nigeria and for in vitro regeneration of this species,

    the attempts of Bais et al. (2002) by standardizing the basal medium produced elite

    plantlets effectively. Similarly, for another species of the same genus, Spilanthes


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  • acmella, a herbal pesticidal plant of north-east India, Purabi Deka and Kalita (2005)

    standardized MS medium for in vitro clonal multiplication and organogenesis.

    In vitro propagation has many advantages over conventional methods of

    vegetative propagation which suffer from several limitations (Murch et al., 2000). With

    in vitro propagation, the multiplication rate is greatly increased and it also permits the

    production of pathogen free propagules. Micropropagation from existing meristems

    yields plants that are genetically identical with the donor plants (Roy et al., 1994). Plant

    regeneration from shoot and stem meristems has yielded encouraging results in

    medicinal plants like Cathranthus roseus, Digitalis purpurea, Dioscorea deltoidea and

    Rauwolfia serpentina. For the biodiesel plant, Jatropha curcus, Kalimuthu et al. (2007)

    developed micropropagation strategies. The nodal explants of this species

    micropropagated successfully when cultured onto the MS medium contained BAP, Kn

    and IAA at 1.5, 0.5 and 0.1mg/l respectively. Somatic embryos were induced directly

    from green cotyledon explants of this species on MS medium fortified with 2 mg/l of

    BAP. Subsequently, the rooting was effectively achieved on MS medium supplemented

    with IAA at 1.0 mg/l.

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

    different medicinal plants. The effect of auxins and cytokinins on shoot multiplication

    of various medicinal plants has been reported. Maragatham and Panneerselvam (2010)

    reported that the combination of auxin, NAA with kinetin at 1.0 and 2.0mg/l

    respectively was effective for callus induction in the medicinal plant, Sida cordifolia.

    Benjamin et al. (1987) reported that 6- benzylaminopurine (BAP) at high concentration

    stimulated the development of axillary meristems and shoot tips in Atropa belladonna.

    Lal et al. (1988) observed a rapid shoot proliferation rate in Picrorrhiza kurroa using

    kinetin at 1.0-1.5mg/l. Direct plantlet regeneration from male inflorescences of

    medicinal yam on MS medium supplemented with 13.94µM kinetin has also been

    reported (Borthakur and Singh, 2002). The highest shoot multiplication of Nothapodytes

    foetida is achieved in medium containing, thidiazuron (TDZ) at the concentration of 2.2

    µM (Ravi, 2002). Similarly, it has been observed that cytokinin is required in optimal

    quantity for shoot proliferation in many genotypes but inclusion of low concentration of

    auxins along with cytokinin triggers the rate of shoot proliferation (Tsay et al., 1989;

    Shasany et al., 1998; Rout et al., 1999a). Wakhlu and Barna (1989) explained that the


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  • production of multiple shoots was higher in Plantago ovata on a medium with 4-6mg/l

    kinetin along with 0.01mg/l NAA. Thidiazuron induced high frequency shoot

    proliferation in Cinerraria marittima reported by Banerjee et al. (2004).

    Xiangquian et al. (2002) reported that high frequency of callus induced by

    11.3µM/l 2, 4-D in rose plant. It has been reported previously that GA3 has induced

    somatic embryogenesis in several rose cultivators (Rout et al., 1991; Marchant et al.,

    1996; Kintzois et al., 1999). Gatica Arias et al. (2010) demonstrated that the effect of

    growth regulators on in vitro regeneration of five cultivars of commercially important

    common bean, Phaseolus vulgaris is significantly varied. They investigated that the

    basal medium with N6 – benzylaminopurine at 5 mg/l and adenine sulphate at 20 or 40

    mg/l resulted in higher average of shoot formation. Trigridia pavonica, an important and

    ornamental medicinal plant of Mexico was successfully regenerated by using tissue

    culture technique by Jose Luis et al. (2010) to meet the demand. In this plant shoot

    formation was determined to be most significant when the twin-scaling explants are

    cultured on MS medium supplemented with 4.5µM 2, 4-dichlorophenoxyacetic acid in

    combination with 2.2 µM BAP. Mir et al. (2010) explained that the shoot multiplication

    was most effective in an endangered medicinal plant, Gardenia gummifera while

    cultured onto the MS medium fortified with BAP at 2.0mg/l. They also reported that the

    combination of BAP and GA3 in the MS medium was effective in shoot proliferation.

    Faria and Illg (1995) reported that the addition of 10µm BA along with 5µm

    IAA or 5µm NAA induces a high rate of shoot proliferation in Zingiber spectabile. They

    have also demonstrated that the number of shoots/explant depends on concentration of

    the growth regulators and the type of genotype used. Nature and condition of explants

    have also been shown to have a significant influence on the multiplication rate of the

    medicinal plant, Clerodendrum colebrookianum (Mao et al.,1995). A simple and

    efficient micropropagation protocol for Vanilla planifolia using shoot tip and nodal

    segments cultured on MS medium containing BA at 1mg/l was reported by Geetha and

    Shetty (2000). As an alternative to the conventional methods of propagation, the plant

    species, Crinum variable was successfully propagated in vitro using twin-scale explants

    (Fennell et al., 2001). They also noted that plant growth regulators were not required for

    the induction of shoots and inclusion of activated charcoal increasing the bulblet size

    and the frequency. High frequency shoot multiplication of Rauwolfia serpentina was


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  • achieved by using nodal explants culturing on MS medium containing 2.5mg/l BA and

    0.1mg/l NAA (Ahmad et al., 2002). Naushaba Baig et al. (2004) have studied the single

    hormonal treatments to understand the requirement of specific type of auxin or

    cytokinin for the propagation of Boerhaavia diffusa through nodal segments and

    explained that it depends upon the endogenous level of the hormones. They

    demonstrated that the nodal explants might be having some cytokinin endogenously in

    higher concentrations which act synergistically with the exogenous application of auxin

    in the medium for in vitro organogenesis. An efficient protocol was developed for high

    frequency plant regeneration from leaf explants of Withania somnifera on MS medium

    supplemented with different concentrations of auxins and cytokinins by Siv

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