Chapter-3 Ri-mediated Genetic Transformation - Shodhganga

96 Pages·2014·1.93 MB·English

Chapter-3

Ri-mediated Genetic Transformation, Secondary
Metabolite Produc tion and Scale-up of Hairy
Roots o f R. serpentina

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3.1 INTRODUCTION
Higher plants represent a valuable resource for a great variety of chemicals or secondary
metabolites which are biologically active compounds and have wide applications in
pharmaceuticals, neutraceuticals, perfumery, cosmetics, flavor, fragrance and pesticide
industries. These often accumulate in specialized tissues, e.g. trichomes at distinct
developmental stages, which makes their extraction, isolation and purification difficult (Kim et
al., 2002b). In many cases organic synthesis of these biologically active compounds is not cost
effective, and extraction from field-grown plants remains the only economical method to
obtain them (Balandrin et al., 1985; Dicosmo and Misawa, 1995). Depending on the plant
species, traditional agricultural methods often require a long period to harvest a crop.
Furthermore, the levels of secondary metabolites in plants are affected by seneral factors,
including geo-climatic, environmental, edaphic, and diseases and pathogens. In addition,
continuously shrinking plant resources and high labor cost involved in the down stream
processes of plant secondary metabolites have hastened the use of plant cell cultures as
potential alternative for the industrial production of such fine and valuable chemicals, since it
is possible to cultivate plant cells, tissues and organs in large quantities in aseptic conditions.
The in vitro technology not only deals with the selection, enhancement and improvement of
the crops but novel pharmacologically active molecules have also been detected, which are not
present in the mother plant. For the industrial application of such in vitro cultured cells two
major questions need to be answered.
• Is the technology feasible?
• Is the economy of the processes competitive with existing production methods?
In the past few years a number of different strategies have been followed in order to improve
the productivity of plant cell cultures. This involves screening and selection of hyper
producing lines and optimization of growth and production media. But genetic instability is the
problem always associated with the cell lines. However, all these strategies are employed only
when a significant level of the compound(s) of interest is present in the cell cultures. Despite
of considerable efforts, only a few processes (e.g. Shikonin, berberine) have been
commercialized using cell cultures (Kieran et al., 1997). The major constraint with secondary
metabolites production from plant cell suspension cultures is that secondary metabolites are
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usually produced by specialized cells and/or at distinct developmental stages (Balandrin et al.,
1985; Mukundan et al., 1997). Some compounds are not synthesized if the cells remain
undifferentiated (Berlin et al., 1985). The undifferentiated plant cell cultures often lose,
partially or totally, their biosynthetic ability to accumulate secondary products (Rokem and
Goldberg, 1985; Charlwood and Charlwood, 1991). This has recently focused attention of
research towards development of other approaches particularly in vitro culture of organized
tissue particularly roots (Balandrin et al., 1985). Normally, root cultures need an exogenous
phytohormone supply and grow very slowly, resulting in poor or negligible secondary
metabolite synthesis.
Genetically transformed roots have been recognized as a new alternative route for enhancing
secondary metabolite production is by roots transformation using the natural vector system
Agrobacterium rhizogenes. A. rhizogenes causes hairy root disease in plants. The technique of
genetic transformation involves the use of the natural ability of the bacterium to transfer genes
in to the host plant genome. The neoplastic (cancerous) roots produced by A. rhizogenes
infection are characterized by high growth rate, genetic stability, short doubling time, ease of
maintenance, growth in hormone free media and ability to synthesize a range of chemical
compounds. Potential of hairy roots for the production of phytochemical such as tropane
alkaloids, indole alkaloids, cardenolides, saponins, terpenoids, flavonoids, chalcones etc.
having high pharmaceutical value have now been established and well documented (Robins,
1998). Hairy roots have also been observed to synthesize novel secondary metabolites, which
are not present in the untransformed tissue (Banerjee et al., 1995).
Large scale culture of hairy roots in bioreactors for the production of phytomolecules at
commercial scale has gained considerable attention over the last few years. Bioreactor capacity
and configuration as well as designing of the culture vessel have been determined depending
upon the type of tissue as well as the location of the product in the plant tissue. A brief
overview of the different types of bioreactors used for the up-scaling of hairy roots of
medicinal and aromatis plants has been described in section 3.2.16.
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The present part of the study was accomplished with the following objectives:
• Selection of the suitable explant in terms of high transformation frequency in response
to two different wild type strains of A. rhizogenes (A4 and LBA 9402)
• Induction and establishment of large number of fast growing hairy root clones
• Assessment of co-relation between the growth and reserpine yield of transformed roots
• Screening and selection of elite root clone(s) exhibiting higher root biomass and/ or
high reserpine yield
• Establishment of a simple protocol for the production of transgenic plants from the
transformed root clone
• Scaling up of reserpine yielding hairy root clone in bioreactor for higher biomass and
secondary metabolite production.

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3.2 REVIEW OF LITERATURE
3.2.1 Hairy roots
The name “hairy root” was first introduced by Steward et al. (1900). Riker et al, (1930) later
described and named the hairy root causing microorganism as Phytomonas rhizogenes, which
was later renamed as Agrobacterium rhizogenes. The first directed transformation of higher
plants using A. rhizogenes was made by Ackermann in 1973 (Ackermann, 1977). A large
number of small, fine, hairy roots covered with root hairs originate directly from the explant in
response to A. rhizogenes infection (Tepfer and Tempe, 1981) and hence the term “hairy root”.
Hairy root cultures of a number of dicotyledonous/ monocotyledonous plants have been
established and found to produce the same secondary metabolites. Hairy root cultures offer
promise for high production and productivity of valuable secondary metabolites in quite a lot
of plants (Srivastava and Srivastava, 2007). The main advantage of hairy roots is that they
often exhibit about the same or greater biosynthetic capacity for secondary metabolite
production as compared to their mother plants (Kim et al., 2002a, 2002b). Plants of a number
of families including Balsaminaceae, Chenopodiaceae, Compositae, Juglandaceae, Labiatae,
Moraceae, Ranunculaceae, Solanaceae, Asteraceae, Cucurbitaceae, Plumbaginaceae,
Apocynaceae, Asclepiadaceae and Umbelliferae have been reported to induce hairy root
disease symptoms on infection with A. rhizogenes (Giri and Narasu, 2000). More than 450
species of many different genera and families are known to be susceptible to the infection by
A. rhizogenes (Hamill and Lidgett, 1997), since then many more additions have been enlisted.
3.2.2 Agrobacterium rhizogenes and Ri T-DNA genes

In nature, the gram negative soil bacterium A. rhizogenes genetically engineers dicotyledonous
plant species into chemical producers of an Agrobacterium food source (opines). Based on the
types of opines produced, the strains of A. rhizogenes can be grouped into five different types:
octopine, agropine, nopaline, mannopine, and cucumopine (Zhou et al., 1998). Agropine
strains are the most often used strains owing to their virulent induction ability. Invasion of A.
rhizogenes inside the plant tissues usually occur at a wounded site. Dicotyledonous plants
produce phenolic compounds particularly acetosyringone and di-hydroxy acetosyringone at
wounded site. Agrobacterium recognizes these signal molecules exuded by susceptible
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wounded plant cells and becomes attached to it (chemotaxis), and subsequently infects the
plant cell at the wounded site. This process leads to the emergence of “hairy roots” at the site
of infection of the plant (Shanks and Morgan, 1999). This activity causes hairy root disease (a
number of small roots emerge as fine hairs at the infection site and proliferate rapidly)
(Balandrin et al., 1985). This phenotypic response (Hairy root) results from the insertion of T-
DNA (transfer DNA) into the plant genome. T-DNA carries a set of genes that encode
enzymes which control auxin and cytokinin biosynthesis. This T-DNA is harboured by the
bacterial Ri-plasmid (root inducing plasmid), which codes for auxin synthesis (Petit et al.,
1983; Ambros et al., 1986), and also contains genes for opine biosynthesis, which are used by
A. rhizogenes as the sole carbon and nitrogen sources for further growth. Products of virulence
(vir) genes located on non-transferrable segment of the Ri plasmid are responsible for excision
of the T-DNA, transfer into the plant cell, and for integration in the genome of the recipient
cell (Giri and Narasu, 2000).

Figure 25: Restriction map of an agropine type Ri –plasmid (pRiA4b). Homology to the octopine type Ti–plasmid
(pTiA6) is indicated by outer most thin bars while homology to the nopaline type Ti–plasmid (pTiT37) is
indicated by the inner bars. Dark regions represent stronger hybridization signal. vir, virulence region; tms, auxin
genes; ori, origin of replication; cT DNA, region of DNA homology between pRiA4b and untransformed N.
glauca (Huffman et al., 1984).
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In the Agropine Ri plasmid T-DNA is referred to as left T-DNA (TL-DNA) and right T-DNA
(TR-DNA) borders. TR T-DNA contains genes homologous to Ti plasmid tumor inducing
genes (Figure-25). Genes involved in Agropine synthesis are also located in the TR DNA
region. T-DNA is transferred to wounded plant cells and it gets stably integrated into the host
genome. Genes encoded in T-DNA are of bacterial origin but have eukaryotic regulatory
sequences enabling their expression in infected plant cells. Synthesis of auxins can be ascribed
to the TR- DNA. However, even in the absence of TR-DNA directed auxin synthesis as in the
mannopine type, which lacks tms loci, root induction occurs. Genes of Ri TL-DNA direct the
synthesis of a substance that recruits the cells to differentiate into roots under the influence of
endogenous auxin synthesis (Ooms et al., 1986; Shen et al., 1988). With the exception of
border sequences, none of the other T-DNA sequences are required for the transfer. Virulence
genes that form the vir region of the Ri plasmid, and chv genes found on bacterial
chromosomes mediate transfer of T- DNA. Transcription of the vir region is induced by
various phenolic compounds released by wounded plant cells such as ‘acetosyringone’ and ‘di-
hydroxy acetosyringone’. Recalcitrant plant species can be transformed by inducing the high
level of vir gene expression (Figure-26) of the bacteria by co-cultivating Agrobacterium with
wounded tissues in media that contains signal molecules and other related supplements
(Stachel et al., 1985; Hu and Alfermann, 1993).

3.2.3 Establishment of hairy roots

A successful transformation event depends upon several factors like strain of A. rhizogenes, an
appropriate explant, age of explant, a suitable antibiotic to eliminate redundant bacteria after
co-cultivation, and a suitable culture medium (Kumar et al., 1991; Giri et al., 1997; Sarma et
al., 1997; Rahman et al., 2004). Many plant parts, such as hypocotyl, leaf, stem, stalk, petiole,
shoot tip, cotyledon, protoplast, storage root, or tuber, have been used to induce hairy roots
(Mugnier 1988; Han et al., 1993; Drewes et al., 1995; Giri et al., 2001; Krolicka et al., 2001;
Azlan et al., 2002). The differences in virulence and morphology can be explained by the
different plasmids harbored by the strains (Nguyen et al., 1992). The bacterial concentration
also plays an important role as sub optimal concentrations may result in lower availability of
bacteria for transforming the plant cells while high concentrations may decrease it by
competitive inhibition (Kumar et al., 1991).

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Figure-26: Activation of vir operons by acetosyringone/ di-hydroxy acetosyringone (A); and Schematic
representation of the production of T-DNA copy (single strand) for transfer in to host plant cells (B)

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Optimization of the nutrient composition for hairy root cultures is a very vital factor to gain a
high production of secondary metabolites. Factors such as the carbon source and its
concentration, the ionic concentration of the medium, phytohormones, the pH of the medium,
light, temperature, and inoculum are known to influence growth and secondary metabolism
(Toivonen et al., 1992; Rhodes et al., 1994; Arroo et al., 1995; Bhadra and Shanks, 1995;
Vanhala et al., 1998; Morgan et al., 2000). Heavy metal ions and the concentrations of
phosphate, nitrate, and ammonia have also been well studied (Payne et al., 1987; Toivonen et
al., 1991; Christen et al., 1992; Sevon et al., 1992). The addition of auxin and elicitors often
increases the levels of secondary metabolites (Dymov et al., 1997; Pitta-Alvarez et al., 1998;
Rijhwani et al., 1998; Singh et al., 1998). Requirement of nutrient condition for an individual
hairy root may be different and therefore should be optimized separately for each species and
for individual clones.

3.2.4 Confirmation of transformation events

The transformation of a plant cell with A. rhizogenes can be confirmed by typical transformed
root morphology exhibited by hairy roots obtained after infection. Since the opine synthesis in
A. rhizogenes infected plant cells is encoded by T-DNA of Ri plasmid (White et al., 1982;
1985), its detection serves as an effective biochemical marker in elucidating the transformed
nature of the cultured root tissue (Petit et al., 1983; Tepfer, 1984; Bakkali et al., 1997; Sasaki
et al., 1998; Bais et al., 2001). Although synthesis of opines is a firm indication that roots are
indeed transformed, the expression of opine genes in hairy root tissue may become unstable
with time (Kamada et al., 1986). T-DNA localization in the host plant genome acts as a
reliable genetic marker to confirm transformation (Mukundan et al., 1997). The techniques to
demonstrate and locate T-DNA incorporation in the host plant chromosomal DNA include
localization of T-DNA by southern hybridization (White et al., 1982; David et al., 1988;
Hamill et al., 1989; Rhodes et al., 1994; Chen et al., 1999; Leljak-Levani et al., 2004). Other
methods include confirmation of the transformed nature of a tissue by screening for the
presence of a foreign gene sequence by DNA “blot dotting” (Draper and Scott, 1988),
localization of T-DNA in plant chromosome tissue by in situ hybridization (Ambros et al.,
1986; Dong et al., 1992), and verification of transgenes as well as determination of changes in
a particular gene sequence resulting from tissue culture by polymerase chain reaction (PCR)
(Dong et al., 1992; Giulietti et al., 1993; Jaziri et al., 1994; Ayadi and Tremouillaux-Guiller,
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2003). The β -glucuronidase (GUS) gene is usually transferred into hairy roots as a reporter
gene and it can be analyzed easily by histological assay (Jefferson et al., 1987; Hosoki et al.,
1994). In some cases, neomycin phosphotransferase II (NPT-II) encoding the kanamycin-
resistance enzyme has been used (Han et al., 1993; Qin et al., 1994). Sometimes, both GUS
and NPT-II are transferred into the hairy roots (Christey et al., 1992; Azlan et al., 2002).
Recently, the gene for green fluorescent protein (GFP) was used successfully as a reporter
gene in Catharanthus roseus L. hairy roots (Hughes et al., 2002).

3.2.5 Characteristics of hairy roots

Hairy roots are characterized by a high degree of lateral branching, profused root branching
and absence of geotropism (Tepfer, 1984). They often grow as fast as or faster than non-
transformed roots due to their extensive branching, resulting in many meristems (Charlwood
and Charlwood, 1991; Flores et al., 1999). Hairy roots generally do not require presence of
phytohormones in the medium (Rao and Ravishankar, 2002). The increase in the number of
branches is approximately logarithmic during the early stages of growth and thus the overall
pattern of growth is similar to cell suspension cultures (Flores and Filner, 1985; Flores, 1986;
Flores and Curtis, 1992). Owing to the highly organized and small-celled region of the
meristem in each lateral, cell cycle times for hairy roots average less than 10 hour (Gould,
1982). Stable integration of Ri T-DNA into host plant genome accounts for the genetic
stability of transformed root cultures. The most important characteristic of transformed roots is
their capability of synthesizing secondary metabolites specific to that plant species from which
they have been developed (Doran, 1989; Flores, 1992). They exhibit biochemical stability that
leads to a high growth rate with a stable and high level of production of secondary metabolites
(Kamada et al., 1986; Aird et al., 1988a, b). Hairy roots are fast growing and plagiotropic and
the plagiotropic characteristic is advantageous as it increases the aeration in liquid medium and
roots grown in air have an elevated accumulation of biomass.

3.2.6 Selection of hairy root lines

Owing to the random integration of T-DNA into the host plant genome, the resulting hairy
roots often show variable patterns of secondary metabolite accumulation. In Duboisia
leichhardtii F. 45 clones of hairy root showed variation in growth rate, alkaloid content, and
productivity among the clones (Mano et al., 1989). Generally, hairy roots are considered to be
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