Chapter 2 Agrobacterium-mediated Genetic Transformation of ...shodhganga.inflibnet.ac.in/bitstream/10603/26730/10... · Agrobacterium -mediated Genetic Transformation of Cenchrus

Chapter 2

Agrobacterium-mediated Genetic Transformation of Cenchrus ciliaris L.

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Agrobacterium-mediated Genetic Transformation of Cenchrus ciliaris L.

2.1 REVIEW OF LITERATURE 2.1.1 Genetic transformation Genetic transformation is one of the ways to change grass genome by introgression of genes from distant or unrelated grass species or other foreign genes. The first step is the development of an efficient regeneration system from embryogenic calli or suspension cultures (Janecek & Ohnoutkova, 2000). For some forage and turf grass species, major advances are achieved in the following areas:

� Establishment of a plant tissue culture method for the competent regeneration of fertile and genetically established plants.

� Generation of transgenic plants through gene gun/ biolistic transformation and direct gene transfer to protoplasts.

� Improvement of intergeneric somatic hybrid grass plants through protoplast fusion. � Development of plant molecular markers for marker assisted selection

Sequencing of expressed sequence tags and the development of deoxyribonucleic acid array technology for gene discovery (Wang et al., 2001). Although, difficulties exist in the genetic manipulation of recalcitrant monocot species, good progress has been made towards the generation of value added novel grass germplasm incorporating traits like improved forage quality. Heeswijak et al., (1994) felt that the event of genetic transformation tools has been slower for monocots than for dicots. Transformation systems have been used to produce transgenic plants of festuca, dactylis etc. for the control of invertebrate pests, virus resistance, improved edibleness and elimination of plant toxins.

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For achieving successful gene transfer, it is necessary to have an efficient method for introducing the desired gene into the cells of the receiver organisms and then a method for growing the transformed cells into fertile transgenic organism. Due to the presence of cell walls, gene transfer in plants cells is more difficult than in bacterial or animal cells. A soil borne tumor inducing phytopathogenic bacterium called Agrobacterium, considered as the natural genetic engineer, has been successfully used for genetic transformation of dicotyledonous as well as monocotyledonous plants (de la Reva et al., 1998). DNA gun has been found to be very effective for gene transfer irrespective of the target cells for plant or animal system (Sanford et al., 1992). Other methods of gene transfer are electroporation (Fromm et al., 1985), PEG-mediated transformation (Uchimiya et al., 1986) and microinjection of DNA (Pena et al., 1987). 2.1.1A Methods of direct gene transfer into plant cells A. Biolistic transformation/ microprojectile or particle bombardment Genetic transformation of cereals is difficult to accomplish and often limited to transient gene expression studies because of the lack of robust regenerative systems and of Agrobacterium to infect recalcitrant cereal tissues. Hence, the method of introducing DNA into cells by physical means (microprojectile bombardment) was developed to overcome the biological limitations of Agrobacterium and difficulties associated with plant regeneration from protoplasts (Sahrawat et al., 2003).

Particle or microprojectile bombardment involves the adsorption of plasmid or linear forms of naked DNA onto the surface of submicron particles of gold, platinum or tungsten driven at high velocity into recipient plant cells using an acceleration device (Sanford, 1988; Sanford et al., 1993) (Figure 2.1). This technology was first reported in the 1980s when the transient activity of genes introduced in such way was found after particle bombardment of maize (Klein et al., 1988), rice, wheat and soybean (Wang et al., 1988) tissues. Southgate et al., (1998) compared four methods for direct DNA transfer in maize genome: tissue electroporation, silicon carbide fibre-mediated gene transfer, tissue electrophoresis, and microprojectile bombardment. The transformation rate was determined by the transient GUS (reporter gene) gene expression after treatment of the immature embryos and subculture of embryogenic calli. High level of

Figure 2.1 BIO-RAD PDS-1000/He Biolistic

the GUS gene expression was obtained in callus tissue cells after micropbombardment and electroporation.

Figure 2.1 BIO-RAD PDS

The biolistic transformation technology is now widely used, and most of the reports on successful transformation of cereals technology, the transgenic plants of the following crops have been obtained: wheat (Triticum aestivum) (Weeks et al., 1993; Becker et al., 1994; Nehra et al., 1994; Sivamani et al., 2000; Liang et al., 2000; Pell(Anderson & Kirihara, 1994; Dennehey et al., 1994; Breusegem et al., 1999; Aulinger et al., 2003), Tritordeumet al., 1994), rice (Oryza sativacereale) (Castillo et al., 1994), oat ((Hordeum vulgare) (Wan & Lemaux, 1994; Manoharan & Dahleen, 2002), and pearl millet (Pennisetum glaucum(Dalton et al., 2003), Paspalum

An essential requirement transformation is pretreatment of explantsto be pre-cultured in the medium containing the osmotic substances M), sorbitol (0.2 M), NaCl (1%) or others. Thetransformation was found before and after the particle bombardment (Vain et al., 1993; Kemper et al., 1996),

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the GUS gene expression was obtained in callus tissue cells after micropbombardment and electroporation.

RAD PDS-1000/He Biolistic Particle Delivery System (www.bio

The biolistic transformation technology is now widely used, and most of the reports on successful transformation of cereals are associated with this method. Using this technology, the transgenic plants of the following crops have been obtained: wheat

) (Weeks et al., 1993; Becker et al., 1994; Nehra et al., 1994; Sivamani et al., 2000; Liang et al., 2000; Pellegrineschi et al., 2001), maize ((Anderson & Kirihara, 1994; Dennehey et al., 1994; Breusegem et al., 1999; Aulinger

Tritordeum, the hybrid of barley and wheat (Hordeum, TriticumOryza sativa) (Li et al., 1993; Christou et al., 1991), rye (

) (Castillo et al., 1994), oat (Avena sativa) (Somers et al., 1992), barley ) (Wan & Lemaux, 1994; Manoharan & Dahleen, 2002), and pearl

Pennisetum glaucum) (Girgi et al., 2006; Latha et al., 2006), Paspalum (Akashi et al., 2002).

requirement for successful gene transfer by means of biolistic transformation is pretreatment of explants material. In the first place, the explants need

cultured in the medium containing the osmotic substances M), sorbitol (0.2 M), NaCl (1%) or others. The maximum frequency of

found when explants were pre-cultured onto the osmotic medium after the particle bombardment (Vain et al., 1993; Kemper et al., 1996),

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the GUS gene expression was obtained in callus tissue cells after microprojectile

1000/He Biolistic Particle Delivery System (www.bio-rad.com)

The biolistic transformation technology is now widely used, and most of the reports on are associated with this method. Using this

technology, the transgenic plants of the following crops have been obtained: wheat ) (Weeks et al., 1993; Becker et al., 1994; Nehra et al., 1994;

egrineschi et al., 2001), maize (Zea mays) (Anderson & Kirihara, 1994; Dennehey et al., 1994; Breusegem et al., 1999; Aulinger

Hordeum, Triticum) (Barcelo al., 1993; Christou et al., 1991), rye (Secale

) (Somers et al., 1992), barley ) (Wan & Lemaux, 1994; Manoharan & Dahleen, 2002), and pearl

Latha et al., 2006), Dichanthium

for successful gene transfer by means of biolistic . In the first place, the explants need

cultured in the medium containing the osmotic substances like mannitol (0.2 frequency of biolistic

ured onto the osmotic medium after the particle bombardment (Vain et al., 1993; Kemper et al., 1996),

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which helped reduce the negative consequences of cell injury. Another main factor for successful gene transfer is the quantity of particles used for bombardment, varied from 30 to 120 mg (Ingram et al., 1999; Brettschneider et al., 1997). The distance between the particle accelerator and the explants is also important (Birch & Franks, 1991). The particles carrying the DNA of must be rapid enough to penetrate cell walls but should not kill the cells. Initially embryogenic suspension cell were used to obtain the transgenic plants through biolistic transformation (Gordon-kamm et al., 1990; Fromm et al., 1990; Register et al., 1994; Kausch et al., 1995). Cells in the suspension culture showed lower extent of aggregation which enabled DNA carrying particles to penetrate into nearly all the target cells. However, the resulting rate of transformation was very low (Klein et al., 1989; Walters et al., 1992). Various plant tissues, such as leaf primordia (Dupuis & Pace 1993) or immature embryos (Brettschneider et al., 1997; O’Kennedy et al., 2001) can also be transformed directly with biolistic methods. For transformation of the genotypes with a low embryogenic potential, the meristematic cells, zygotic embryos and immature embryos are used (Lowe et al., 1995; Zhang et al., 1996; Songstad et al., 1996).

The transformation efficiency of the immature embryos of maize achieved by Koziel et al., (1993) was 1%. Some workers could optimize the biolistic transformation method to achieve the transformation rate as high as 3% (Brettschneider et al., 1997). Rasmussen et al., (1994) simplified the transformation method by treatment of Escherichia coli cells with 1% phenol and mixing them with microparticles of tungsten, which were then used for transformation instead of isolation of plasmid DNA. The transformation of the cell suspension culture with this procedure resulted in thousands of cells with transient expression but only six cells with constant expression of introduced genes was recorded. The nonstop progress of biolistic transformation devices has enabled the workers to reduce the time required and raise the effectiveness of particle microbombardment (Vain et al., 1993; Pareddy et al., 1997). The standard efficiency of transformation of numerous plants by the biolistic way varies from 1 to 3%. In some cases with appropriate standardization of the machinery, 18.1% of transformation rate has also been reported (23 transgenic plants were found out of 127 calli); though, not all of the transformed plants expressed the introduced genes (Tang et al., 2000).

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The disadvantage of this technology is that the number of inserted copies of introduced genes in one cell cannot be controlled. A lot of copies of the same gene get integrated into the plant genome of the same cell which usually results in gene silencing. Very often, sequences of the introduced genes are incomplete, which is also unwanted (Walters et al., 1992; Brettschneider et al., 1997). An additional disadvantage of this technique is the limitation on the size of introduced DNA fragment. So far, the researchers have succeeded in biolistic transfer of the DNA fragments upto 10 Kb (Register et al., 1994; Tang et al., 2000). This method can also lead to the emergence of chimeric plants. In addition, lack of control over the velocity of bombardment result into tissue damage. In spite of all the above-mentioned disadvantages, the biolistic method is now the most popular method for direct DNA transfer in cereal crops.

B. Transformation of protoplasts One of the most primitive procedures of direct foreign gene transfer into plant cells were based on protoplasts. Protoplasts are plant cells without the cell wall and therefore, very appropriate for genetic manipulations. The machinery has no limitations about the species of plants to be transformed; the main restrictions are procedure for isolation and in vitro culturing protoplasts and their further regeneration into plants. Poly Ethylene Glycol solution at alkaline pH, containing Ca++ ions is added to assist foreign DNA penetration through the plasma membrane (Negrutiu et al., 1987). For the period of PEG treatment, the plant-cell membranes are distorted by surface-tension forces owing to the cell density differences between the PEG solution and the protoplast. The alkaline medium moderately damages the plasma membranes, whereas the Ca++ ions stabilize the newly formed membrane structures. After such treatment, as a result of pinocytosis the DNA molecule possibly gets transported into the plant cell. Using protoplast transformation method, the first transgene cell lines of several cereal crops were obtained, including the single grain wheat (Lorz et al., 1985; He et al., 1994); maize (Faranda et al., 1994; Rhodes et al., 1988; Golovkin et al., 1993); rice (Toriyama et al., 1988; Shimamoto et al., 1989; Datta et al., 1990) and barley (Lazzeri et al., 1991; Nobre et al., 2000). The efficiency of the plant protoplasts transformation was not higher than 0.01 to 0.1%, yet, in some cases it was found as high as 10% (Wang et al., 2000).

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However, the direct transformation of protoplasts method with target DNA had few disadvantages as well, the most important of them being difficulties in extracting protoplasts and their regeneration. Furthermore, there is a high chance of getting somaclonal variations, However, this protocol is commonly used for transient expression of genes for the assessment of different constructs, for example, to identify the regulatory elements of plant gene promoters (Kuchuk, 1997, Zinkevich et al., 1988; Bogdarina et al., 1993). C. Electroporation Electroporation-mediated transformation requires the application of strong electric field pulses to cells and tissues which causes some type of structural rearrangement of the cell membrane. It is a modification of the direct protoplast treatment technology and is highly reproducible. The technique is based on the treatment of protoplasts with electric pulses of high voltage from 200 to 1500 V/cm for short period like dozens of microseconds to dozens of milliseconds in the existence of the foreign DNA (Bates, 1995; Fromm et al., 1985). By this method, a wide range of explants can be transformed but the main disadvantage of this technology is high rate of destruction of the foreign DNA constructs caused by electric pulses resulting in very low transformation rate. Zhang et al., (1988) were the first to obtain transgenic rice plants using this machinery. By way of electroporation, the target DNA can be transferred into protoplasts, plant meristematic tissues and embryos (Akella & Lurguin, 1993). Dillen et al., (1995) obtained blue spots in tissues of the meristem and embryos of electroporated cereals, which confirmed their successful transformation (using uidA gene). Similarly, foreign DNA was successfully introduced into the rice embryos (Tada et al., 1990), maize (Songstad et al., 1993) and the scutellar cells of wheat embryos (Kloti et al., 1993; He & Lazzeri, 1998) using electroporation. The transformation efficiency of 0.4% under voltage 750 V/cm was recorded in immature embryos of wheat by Sorokin et al., (2000). This technique can be used for obtaining the transgenic plants of genotypes with a low morphogenic potential. Leedell et al., (1997) were able to obtain transgenic plants after treating isolated maize pollen grains with electric pulses in the presence of plasmid DNA carrying the GFP gene and subsequently fusing with the ovule cells in calcium-

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containing medium. This technology can be used for obtaining the transgenic plants for genotypes with a low morphogenic potential. D. Silicon carbide-mediated transformation (SCMT) SCMT is one of the most complex methods of plant transformation. Silicon carbide fibers are basically added to a suspension containing plant tissue (cell clusters, embryos, calli) and plasmid, and then mixed by vortexing. Plasmid DNA-coated fibers go through the cell wall through small holes created as results of collision between the plant cells and silicon carbide fibers (Kaeppler et al., 1990; 1992; Wang et al., 1995). This technique was used successfully to obtain transgenic maize and wheat plants transformed with the neomycin phosphotransferase II, phosphinotricine acetyltransferase, and ß-glucuronidase genes (Kaeppler et al., 1992; Frame et al., 1994; Thompson et al., 1995; Petolino et al., 2000; Briside et al., 2000). There are several known examples of deriving transgenic from cell colonies or plants in maize (Bullock et al., 2001; Wang et al., 1995), rice (Nagatani et al., 1997), Wheat (Serik et al., 1996), Lolium multiflorum, Lolium perenne Agrostis stolonifera, Festuca arundinacea, and (Dalton et al., 1997) by SCMT. This technique continues to be used for transformation of cereal crops, however, its main disadvantage is that only the embryonic cells will be used for transformation, therefore limiting the quantity of properly developed plants. E. Transformation through pollination The requirement to use in vitro cell cultures for transformation is often difficult to make fertile transgenic plants for the majority of the cereals. One promising solution is the use of a method based on the natural process of pollination. Ohta, (1986) and Chesnokov & Korol, (1993) used this technology for the foreign DNA transfer during the pollination. Pena et al., (1987) tried to obtain transgenic rye seeds by direct injection of target DNA into the young floral buds 14 days before meiosis. The transformation methodology via pollen-tube pathway has vast scope in agriculture (Song et al., 2007). Foreign DNA is applied to cut styles shortly after pollination and DNA reaches the ovule by flowing down the pollen-tube. This method, alleged pollen-tube pathway

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(PTP), was practically applied first time for the genetic transformation of rice (Luo & Wu, 1988) and then in wheat (Mu et al., 1999). The authors found transgenic plants at extremely high frequency. However, transformation during pollination/fertilization has not been widely accepted for cereal crops because of reduced seed yield, low transformation rate, requirement of much time and efforts, and seasonal dependence of the technique. 2.1.1B The indirect gene transfer using Agrobacterium as vector into

plant cells A. Agrobacterium-mediated tansformation In this method, bacteria namely, A. tumefaciens or A. rhizogenes are employed to introduce foreign genes into plant cells. Agrobacterium tumefaciens is a soil-borne gram (-) bacterium, which might induce disease so called crown gall in plants by transferring a distinct portion of its DNA (Chilton, 1977; Zambryski, 1983; Klee and Rogers, 1989; Binns and Thomshow, 1988). The two main regions essential are; oncogenic T-DNA and the virulence genes, that encode proteins for T-DNA transfer. T-DNA transfer is reliant on direct repeat and flanking sequence of 25basepair. The T-DNA is capable of inducing tumours in transformed plant, accompanied by products of 3 genes, which code for an auxin (Schroder et al., 1984; Thomshow et al., 1984), cytokinin (Akoyoski et al., 1984) and opine compound. A. tumefaciens plasmid transfers only T-DNA. In hypothesis, it is possible to introduce any gene into plant genome (Zambryski et al., 1983). The T-DNA region is deleted from Ti plasmid to forestall the over production of phytohormone, that interferes with normal growth of plant development. The disarmed strain as it is known has vir region. These vir genes are crucial for T-DNA transfer and organized into a number of operons (virA, virB, virC, virD, virE, virF, virG, and virH) on the Ti-plasmid and other operons (chvA, chvB, and chvF) that are chromosomal. The machinery of gene transfer from Agrobacterium to plant cells involves many steps, which consist of bacterial colonization of T-DNA transfer complex, T-DNA transfer, and integration of the T-DNA into the whole plant genome (Figure 2.2).

Figure 2.2 Schematic diagram of the or within the bacterium (chemical signaling, vir gene induction, and Tthe plant cell (bacterial attachment, Thighlighted, along with genes and/

The progression of T(phenolic compounds) receDNA transfer, presumably due to induction by phenolic compounds produced during cell repair or throughout the formation of new cells. In reaction, a signal received by virA activates a cascade of oproteins nick equally the left and right borders on the base strand of Tensuring T-DNA molecule jointly with many vir cytoplasm of host cell through a channel fashioned by the VirB protein complex (T-strand conjugate is probably coated by VirE2, making the Tsingle-stranded DNA-binding cell, wherever it presumably functions to protect the Tcomplex then enters the nucleus by a import machinery of the host cell. This facilitates integration of the Thost cell genome at random positions by a method of nonhomologous, or more specifically, illegitimate recombination.

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Figure 2.2 Schematic diagram of the Agrobacterium infection process. Critical steps that occur to or within the bacterium (chemical signaling, vir gene induction, and T-DNA processing) and within the plant cell (bacterial attachment, T-DNA transfer, nuclear targeting, and Thighlighted, along with genes and/ or proteins known to mediate these events

The progression of T-DNA transfer is initiated upon receipt of specific signals (phenolic compounds) received from host cells. Wounding also additionaly promotes TDNA transfer, presumably due to induction by phenolic compounds produced during cell repair or throughout the formation of new cells. In reaction, a signal received by

activates a cascade of other vir protein machinery genes. Later, proteins nick equally the left and right borders on the base strand of T

DNA molecule jointly with many vir proteins then is exported into the cytoplasm of host cell through a channel fashioned by the AgrobacteriumVirB protein complex (Christie, 1997). Before its access into the cytoplasm, the VirD2strand conjugate is probably coated by VirE2, making the T-complex. VirE2 is a

binding Agrobacterium protein that’s transported into the plant cell, wherever it presumably functions to protect the T-DNA from degradation

x then enters the nucleus by a dynamic mechanism mediated by import machinery of the host cell. This facilitates integration of the Thost cell genome at random positions by a method of nonhomologous, or more specifically, illegitimate recombination.

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Critical steps that occur to

DNA processing) and within and T-DNA integration) are

known to mediate these events (Gelvin, 2000).

DNA transfer is initiated upon receipt of specific signals ived from host cells. Wounding also additionaly promotes T-

DNA transfer, presumably due to induction by phenolic compounds produced during cell repair or throughout the formation of new cells. In reaction, a signal received by

protein machinery genes. Later, virD1 and virD2 proteins nick equally the left and right borders on the base strand of T-DNA. The

then is exported into the Agrobacterium VirD4 and

Before its access into the cytoplasm, the VirD2-complex. VirE2 is a

protein that’s transported into the plant DNA from degradation. The T-

dynamic mechanism mediated by the nuclear import machinery of the host cell. This facilitates integration of the T-strand into the host cell genome at random positions by a method of nonhomologous, or more

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The fact that an element of the Ti plasmid was transferred and integrated into the plant genome throughout tumor formation, recommended that the plasmid could be used as a vector to transfer alternate genes. Thus, strategies were developed to insert gene into the Ti plasmid (van Haute et al., 1983; de Framond et al., 1983; Hoekema et al., 1983). Main advantages of the Agrobacterium- mediated transformation technology is to facilitate the DNA transfer method implicated as totally natural source and does not need destruction of plant cells in transformation. One more advantage is that the T-DNA is delivered instantly into the cell nucleus, and this significantly increases the chance of integration of target genes into the plant cell genome and also introduces a small number of copies of foreign DNA per cell as compared with other transformation methods.

However, efficient methodologies of Agrobacterium-mediated gene transfers have been established mainly for dicotyledonous plants. Monocot plants have been considered to be outside the Agrobacterium host range. Further discoveries in this field indicated that even in cereals and non-host species, Agrobacterium-mediated DNA transfer system could work with the aid of phenolic compound called acetosyringone (Stachel et al., 1985; Schafer et al., 1987) or by using specific Agrobacterium strain or vector (Hiei et al., 1994). The vir genes can also be stimulated by few monosaccharides, such as myo- inositol and D-galactose, L- arabinose and others; via low pH and at optimal temperature of 22°C (Ankenbauer & Nester, 1990; Dillen et al., 1997). It was reported that the effectiveness of transformation through Agrobacterium depends on the age and physiological status of the host plant (Zakharchenko et al., 1999) as well as on the present phase of the cell cycle of the transformed cell (Kausch et al 1995; Villemont et al., 1997). The co-culturing period of explants to the Agrobacterial cell suspension and the duration of co-cultivation in antibiotic-free media also play major role in transformation. The composition of media being used both for co-culturing and for consequent co-cultivation of explants is also very essential (Jones et al., 2005).

So many factors influencing Agrobacterium-mediated transformation of cereals consist of plant genotype, type of explant, Agrobacterium strain, binary vector, and extensive variety of inoculation/ cell density and co-culture environment. Antinecrotic treatments (like AgNO3), using antioxidants and bactericides (cefotaxime/ timentine), osmotic treatments, desiccation of explants before and after Agrobacterium infection,

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inoculation and co-culture media compositions have all been shown to be essential for recovering transgenic cereals (Cheng et al., 2004). Beyond any doubt, the event of plant transformation procedures based on Agrobacterium mediated gene transfer for major species are valuable and therefore the results obtained in recent years indicate a promising future. 2.1.2 Regeneration of transgenic plants Embryogenic calli are generally used as explants for genetic transformation of cereal plants. In general, success of transformation wholly depends on the plant regeneration potential of the transformed explants. Measures to increase the regeneration potential of crops are being considered throughout the world. New ways and approaches are being tried to widen the length of transformable crops and raise the plant regeneration capacity of transformed explants. In majority of cases, the regeneration capacity depends on genotype, explant and the composition of medium (Rakoczy-Trojanowsk & Malepszy, 1995; Fennell et al., 1996; Rout & Locas, 1996; Redway et al., 1990; Bahieldin et al., 2000; Eudes et al., 2003). For induction of morphogenesis in vitro, natural phytohormones and synthetic hormone-like regulators are used which consist of auxins such as IAA, 2,4-D in varying combinations with cytokinins like BA/ kinetin (Huang & Wei, 2004). The cefotaxime, was verified to be effective in regeneration from crop explants. Now a day, cefotaxime is generally used for removal of Agrobacterium from explants during genetic transformation. Mathias & Boyd, (1986) and Mathias & Mukasa, (1987) reported that cefotaxime induced callus growth and improved the organogenesis of wheat and barley plants. After cefotaxime treatment, Borrelli et al., (1992) observed the rising rates of embryogenic callus induction, plant regeneration of durum wheat, and increase in the number of regenerated plants. Parallel results were also noticed by Rao et al., (1995) when sorghum in vitro raised cultures were treated with cefotaxime. Cefotaxime was reported to accelerate the callus development stage and plant regeneration rate by a factor of 3 to 18, depending upon the plant genotype (Danilova & Dolgikh, 2004). The cephalosporin family antibiotics are very effective when applied in comparatively low concentrations of 50 to 150 mg/l. They are characterized through broad spectrum of biological activity and low toxicity in relation to plants. Other inducers of morphogenesis are a few amino acids and

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oligosaccharides (Eudes et al., 2003; Dolgikh et al., 1998). For example, xyloglucan stimulated the morphogenesis of wheat cultures (Pavlova et al., 1992). Another example is emistim which helped increase the number of shoots regenerated from maize calli (Dias & Dolgikh, 1997). 2.1.3 Analysis of transgenic plants The selection of transgenic plants is a major step in transformation process. The DNA to be integrated into the transformed cell is normally a combination of target foreign genes and reporter genes or selectable marker that helps to differentiate between the transformed and non-transformed cells/ tissues. Two types of genes are used for testing the putative transgene plants:

1. The selectable marker genes to make the plant/ cell resistant against antibiotics or herbicides.

2. The reporter genes that help to simply distinguish the expression of the integrated genetic constructs. nptII gene of E. coli is generally used to make plants resistant to an antibiotic kanamycin and hence works as a selectable marker for genetic transformation. This nptII gene is very frequently used for plant transformation of several species of monocots and dicots plants, but generally used for transformation of monocotyledonous plants, in about 33% (Miki & McHugh, 2004). Hph or, hpt genes isolated from E. coli coding for the enzyme hygromycin phosphotransferase (Gritz & Davie, 1983) confer resistance to hygromycin, and has been used by many workers in maize (Walters et al., 1992), pearl millet (Lambe et al., 2000; 1995) and sorghum (Hagio et al., 1991), buffel grass (Bhat et al., 2001). The bar gene of Streptomyces hygroscopicus (Thompson et al., 1987) which make the transformed plants resistant to bialophos is now broadly used as selectable marker gene in cereal transformation. In addition, the EPSP and glyphosate oxidase (GOX) genes are used to make plants resistant to the glyphosate, the herbicide (commercially available as Roundup) (Miki & McHugh, 2004). All the genes have been successfully used for genetic transformation of cereals like wheat, rice, and maize (Gordon-kamm et al., 1990; Fromm et al., 1990; Rathore et al., 1993; Vasil et al., 1992; Zhou et al., 1995; Howe et al., 2002).

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Reporter genes are used to identify and evaluate the expression of genes in the transgenic plants. The GUS (uidA) gene originating from E. coli is commonly used for genetic transformation of plants (Vain et al., 1993a; Leduc et al., 1994; Girgi et al., 2002; Devi & Sticklen, 2002; Goldman et al., 2003). Through histological staining, one can examine the expression of GUS gene in transformed tissue. Luciferase (LUX) gene (Ow et al., 1986) known as a reporter gene in rice (Baruah et al., 1999) and barley (Harwood et al., 2002) which is isolated from firefly and analyzed by using chemiluminescence procedure. An additional reporter gene being used for genetic transformation of cereals crops is GFP, initially cloned from the Aequorea victoria (jellyfish) (Prasher et al., 1992). The increasing interest for this gene is due to the distinctive property of the GFP protein to release the fluorescent light within the visible range of spectrum when exposed close to UV beam. No treatment of any kind is needed to identify the expression of GFP gene, thus allowing for diverse types of research and analyses to be carried out with plants. So many other marker genes have been reviewed by Zvereva & Romanov, (2000) and Miki & McHugh, (2004) .

After selection of plants carrying the selectable marker and reporter genes, molecular analysis is completed to validate the presence of the genes in transformants. The polymerase chain reaction (PCR) is commonly used for genetic transformation. Still, when analyzing the T0 lines, one could obtain fake results caused by the Agrobacterium infection of untransformed plants. To avoid confusion, further PCR test can be carried out with the unique gene specific primers to see if the later are present in tested plants. Southern hybridization methods are used for more reliable confirmation for the presence of the target foreign genes in the transformed plants and also to assess the number of copies of the target gene in the whole plant genome. This method is still being used successfully. Using Real Time PCR tools for confirming transformation is now coming into wide perform. 2.1.4 Genetic transformation of apomictic Cenchrus ciliaris The genetic transformation of Cenchrus was first done by Ross et al., (1995) through microprojectile bombardment (biolistic method) where mature embryos were used as the target tissue. In 2001, Bhat and his coworkers optimized the biological and physical

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parameters for particle inflow gun mediated genetic transformation. They bombarded plasmid DNA carrying the actin promoter coated on 1.6µ gold particles at a helium pressure of four bars from a distance of 10 cms for 10µ seconds and 28mm Hg vaccin the chamber and produced successful transient GUS expression in the targeted calli. Actin promoter has been found to be more efficient in driving expression of the GUS gene in buffel grass, followed by ubiquitin and CaMVother food crops, there have been very few reports on forage grasses like development of efficient and reproducible transformation protocol is a necessity to understand key developmental events like apomixis in this species.

Table 2.1 Studies on genetic transformation of

2.2 MATERIAL AND METHODSPlant Material In this study, mature seeds of Agrobacterium-mediated transformation. 2.2.1 Isolation of shoot apicesThe seeds were manually dehusked and surface sterilized using 70% (v/v) ethanol for 1 min, followed by 5 min in 0.1% (w/v) mercuric chloride. The seeds were then washed 4-5 times with sterile distilled water to remove the traces of mercuric chloride and then soaked overnight in 100ppm solution of Gibberellic Acid (GAsynchronize germination. The treated seeds were germinated under aseptic conditions on MS medium with 1mg/l TDZ in light for 2shoot apices, comprising of shoot apical meristem and a part of mesocotyl were excised

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parameters for particle inflow gun mediated genetic transformation. They bombarded plasmid DNA carrying the actin promoter coated on 1.6µ gold particles at a helium pressure of four bars from a distance of 10 cms for 10µ seconds and 28mm Hg vacc

successful transient GUS expression in the targeted calli. Actin promoter has been found to be more efficient in driving expression of the GUS gene in buffel grass, followed by ubiquitin and CaMV-35S promoters. In comparison to

od crops, there have been very few reports on forage grasses like Cenchrusdevelopment of efficient and reproducible transformation protocol is a necessity to understand key developmental events like apomixis in this species.

tic transformation of Cenchrus ciliaris

MATERIAL AND METHODS

In this study, mature seeds of Cenchrus ciliaris cv. IG-3108 were used for mediated transformation.

hoot apices dehusked and surface sterilized using 70% (v/v) ethanol for 1

min in 0.1% (w/v) mercuric chloride. The seeds were then washed 5 times with sterile distilled water to remove the traces of mercuric chloride and then

00ppm solution of Gibberellic Acid (GA3) to break dormancy and synchronize germination. The treated seeds were germinated under aseptic conditions on MS medium with 1mg/l TDZ in light for 2-3 days at 25ºC. Emerging 2

hoot apical meristem and a part of mesocotyl were excised

parameters for particle inflow gun mediated genetic transformation. They bombarded plasmid DNA carrying the actin promoter coated on 1.6µ gold particles at a helium pressure of four bars from a distance of 10 cms for 10µ seconds and 28mm Hg vaccum

successful transient GUS expression in the targeted calli. Actin promoter has been found to be more efficient in driving expression of the GUS

promoters. In comparison to Cenchrus and

development of efficient and reproducible transformation protocol is a necessity to

3108 were used for

dehusked and surface sterilized using 70% (v/v) ethanol for 1 min in 0.1% (w/v) mercuric chloride. The seeds were then washed

5 times with sterile distilled water to remove the traces of mercuric chloride and then ) to break dormancy and

synchronize germination. The treated seeds were germinated under aseptic conditions 3 days at 25ºC. Emerging 2-3 mm long

hoot apical meristem and a part of mesocotyl were excised

from 2-3 days old seedling using a sterile scalpel blade and cultured horizontally on solid MS medium with 3 mg/l TDZ for 1 day at 25ºC under 16 h photoperiod (40µmolPre-cultured shoot apicetransformation. 2.2.2 AgrobacteriumAgrobacterium tumefaciensand pCAMBIA 1305.1 was used in our experimentspCAMBIA 1301 and 1305.1 contains GUS gene with a modified castor bean catalase intron as a reporter and hptII genemarker driven by CaMV 35S promoter.

Figure 2.3 Schematic diagram of Tselected for Southern Hybridization for pCAMBIA 1301 has also been indicated.

A

Figure 2.4 Map of Binary Vectors (A)

63

3 days old seedling using a sterile scalpel blade and cultured horizontally on solid MS medium with 3 mg/l TDZ for 1 day at 25ºC under 16 h photoperiod (40µmol

cultured shoot apices of 5-6 mm were used as explant for Agrobacterium

Agrobacterium strain and plasmid tumefaciens strain EHA 105, containing binary vector

and pCAMBIA 1305.1 was used in our experiments (Figure 2.3-2.4)pCAMBIA 1301 and 1305.1 contains GUS gene with a modified castor bean catalase

as a reporter and hptII gene conferring resistance to Hygromycin as a selection marker driven by CaMV 35S promoter.

Figure 2.3 Schematic diagram of T-DNA region of pCAMBIA 1301 and 1305.1. The region of probe selected for Southern Hybridization for pCAMBIA 1301 (GUS) and PCAMBIA1305.1

A B

Figure 2.4 Map of Binary Vectors (A) pCAMBIA 1301, (B) pCAMBIA1305.1 (www.cambia.org)

Chapter 2

3 days old seedling using a sterile scalpel blade and cultured horizontally on solid MS medium with 3 mg/l TDZ for 1 day at 25ºC under 16 h photoperiod (40µmol-2s-1).

Agrobacterium mediated

binary vectors pCAMBIA 1301 2.4). The T-DNA of

pCAMBIA 1301 and 1305.1 contains GUS gene with a modified castor bean catalase conferring resistance to Hygromycin as a selection

DNA region of pCAMBIA 1301 and 1305.1. The region of probe

and PCAMBIA1305.1 (hygromycin)

B

(www.cambia.org)

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64

YENB medium which contains yeast extract (7.5 mg/l) and nutrient broth (8 mg/l) [pH 7.5] was used for the growth of Agrobacterium. The medium was supplemented with Kanamycin (50 mg/l) and Rifampicin (25 mg/l).

A single colony of Agrobacterium strain was grown overnight in small culture (10 ml) containing liquid YENB medium supplemented with 25 mg/l rifampicin and 50 mg/l kanamycin antibiotic at 28°C with shaking at 180 rpm. From overnight small culture, 1 ml was resuspended in 50 ml medium containing 200 µM acetosyringone specified O.D600nm is approximately 0.1 and then was grown for 2-3 hours beneath same conditions till an O.D600nm of 0.8-1.0 was reached. The agro-suspension was centrifuged at 6000 rpm for 10 min at 4°C. The pellet was later resuspended in MS-inf medium to be used for co-culturing. 2.2.3 Hygromycin sensitivity test Hygromycin is the most accepted selectable marker gene used in plant transformation vectors. For successful selection, the non transformed plant cells must be susceptible to antibiotics. In this experiment hygromycin sensitivity was tested to determine the threshold concentration of hygromycin required to be used for the selection of the transformants. Shoot apices were cultured on medium (MS + 3 mg/l TDZ) containing various levels of Hygromycin (0, 5, 10, 15, 20, 25, 30 & 50 mg/l) and lethal dose was determined 2 times after at 15 days interval. The concentration of Hygromycin lethal for the explants was used in ensuant transformation experiments. 2.2.4 Transformation of shoot apices by Agrobacterium tumefaciens After one day incubation, shoot apices explants were immersed in MS–inf medium and kept for different duration ranging from 10-60 minutes with gentle shaking (80 rpm at 25ºC). Before agro-infection, the O.D600nm of Agrobacterium strain was adjusted from 0.6 to 2.0 in MS-inf medium (Table 2.2). To assess the effect of negative pressure, (0.5 x 105 pa) vacuum pump was used throughout agro-infection for 30 minutes. The explants were taken out of bacterial suspension and were blot dried using sterile blotting paper to get rid of excess Agrobacterium and then placed on co-cultivation medium for 1-6 days. Co-cultivation was conceded out at 25°C in 16 hours light on semi-solid

Chapter 2

65

medium. Various concentrations of acetosyringone (0-500 µM) were tested in co-cultivation medium. After co-cultivation, shoot apices were washed with MS salts containing 250 mg/l of cefotaxime or sterile water containing cefotaxime, blot dried with sterile blotting paper and subcultured to recovery medium for one week to reduce the growth of Agrobacterium and then transferred onto the first selection medium for 30 days at a subculture time of 15 days to stimulate the production of transgenic multiple shoots. After hygromycin selection, surviving shoots were transferred onto pre-regeneration medium for 5-6 weeks. Finally, these multiple shoots were regenerated on regeneration medium for 2-3 weeks and were rooted on rooting medium. After 20 days, rooted plants were hardened and transferred to small cups with sterile vermiculite. 2.2.5 Agrobacterium-mediated transformation of embryogenic calli Embryogenic calli as explant of genotype IG-3108 were used for Agrobacterium mediated transformation. Calli were induced by immature inflorescence, on MS + 3 mg/l 2,4-D and maintained on MS + 3 mg/l 2,4-D + 0.5 mg/l BA+ 400 mg/l Proline+ 400 mg/l glutamine + 300 mg/l casein hydrolysate. Single colony each of Agrobacterium strain EHA 105 containing construct pCAMBIA 1301 and 1305.1 was grown on liquid 10 ml of AB medium [AB buffer composition = 6 g K2HPO4, 2.6 g NaH2PO4.2H2O; AB salt = 2 g NH4Cl, 0.6 g MgSO4.7H2O, 0.3 g KCl, 0.3g CaCl2.2H2O, 0.005 g FeSO4.7H2O and 5 g D-glucose) (pH 7.)] supplemented with 25 mg/l rifampicin and 50 mg/l kanamycin at 28°C with gentle shaking of 180 rpm. Overnight grown culture was resuspended in large AB medium with same condition for 2-3 hours such that O.D600nm is around 0.1. The suspension culture was pelleted down at 6000 rpm for 10 min at 4°C. The pellet was later resuspended in MS-inf medium to an O.D600nm of 1.0 and calli was immersed in it for 30 minutes under vacuum treatment, then those calli were co-cultivated for 3 days on co-cultivation medium (MS + 3 mg/l 2,4-D+ 0.5 mg/l BA). Embryogenic calli were washed with sterile distilled water for 2-3 times, and after that washed with cefotaxime solution (250 mg/l) for 5 minutes, finally blot dried and subcultured on recovery medium for one week. After recovery phase, calli were transferred to selection medium containing 30 mg/l hygromycin for 30 days at 15 days subculture intervals. After 30 days of selection medium, induced calli were transferred on pre-regeneration medium.

Chapter 2

Table 2.2 Composition of agro culture media used in agroselection, regeneration and rooting.

2.2.6 Histochemical GUS assay Six days after transformation, histochemical GUS assay was performed according to the method developed by Jefferson, (1987), in order to observe transient GUS expression. For GUS assay, explants were chloro-3-indolyl-β-D-glucuronic acid (Xwere vacuum infiltrated at 600 mmHg for 1solution by the plant tissue. Then the explants were incubated oSubsequently, the tissues were cleared in 70% ethanol at 25°C in dark condition. At the end of the incubation period, the explants were examined under stereomicroscope and scored for the number of explants showing blue stain per treatment 2.2.7 Statistical analysis Each experiment was replicated 3 times and each set within a replicate consisted of at least 60 explants. The transformation frequency was calculated as the total number of shoot apices expressing GUS gene per total number of explants infected by Agrobacterium x 100. For all the experiment, statistical analyses were carried out by using SPSS Software version 16. Analysis of variance (ANOVA) and Duncan multiple

66

Table 2.2 Composition of agro culture media used in agro-infection, co-cultivation, recovery, selection, regeneration and rooting.

Histochemical GUS assay Six days after transformation, histochemical GUS assay was performed according to the

by Jefferson, (1987), in order to observe transient GUS expression. For GUS assay, explants were immersed in 1 mM chromogenic substrate 5

glucuronic acid (X-Gluc) as substrate in falcon tubes and they were vacuum infiltrated at 600 mmHg for 1-2 minutes for better absorption of substrate solution by the plant tissue. Then the explants were incubated overnight at 37°C. Subsequently, the tissues were cleared in 70% ethanol at 25°C in dark condition. At the end of the incubation period, the explants were examined under stereomicroscope and scored for the number of explants showing blue stain per treatment.

experiment was replicated 3 times and each set within a replicate consisted of at

transformation frequency was calculated as the total number of expressing GUS gene per total number of explants infected by

all the experiment, statistical analyses were carried out by using SPSS Software version 16. Analysis of variance (ANOVA) and Duncan multiple

cultivation, recovery,

Six days after transformation, histochemical GUS assay was performed according to the by Jefferson, (1987), in order to observe transient GUS expression.

substrate 5- bromo-4-Gluc) as substrate in falcon tubes and they 2 minutes for better absorption of substrate

vernight at 37°C. Subsequently, the tissues were cleared in 70% ethanol at 25°C in dark condition. At the end of the incubation period, the explants were examined under stereomicroscope and

experiment was replicated 3 times and each set within a replicate consisted of at transformation frequency was calculated as the total number of

expressing GUS gene per total number of explants infected by all the experiment, statistical analyses were carried out by

using SPSS Software version 16. Analysis of variance (ANOVA) and Duncan multiple

Chapter 2

67

range test (DMRT) were used to investigate the statistical significance, and the significance of differences among means was carried out using LSD at a significance level of P = 0.05. 2.2.8 Molecular analysis 2.2.8.1 Confirmation of presence of transgene To confirm the presence of transgene, the control and the putatively transformed plants were tested for the presence of hptII and GUS gene by PCR using specific primers and Southern blotting. 2.2.8.1.1 Isolation of plasmid DNA from Agrobacterium culture Plasmid DNA from Agrobacterium cultures was isolated using HiYield™ Plasmid Mini Kit (Real Genomics, Cat.No. YPD100), following the manufacturer’s protocol and used as control in PCR analysis. Plasmid DNA was eluted in elution buffer (30 µl) then checked by running on 0.8% agarose gel. 2.2.8.1.2 Genomic DNA extraction Genomic DNA was extracted using Qiagen DNeasy Plant Maxi Kit as per manufacturer’s protocol. 1g of young leaves of transgenic plants (T0) and control plants were collected and then followed the method same as given in kit. 2.2.8.1.3 PCR analysis of control and transgenic plants Polymerase chain reaction was performed using DNA extracted from control and transformed plants to confirm successful transformation protocol. The reaction was set up using GUS and Hygromycin specific primers listed in Table 2.3. The reaction set up and cycling conditions used are as follows:

a. PCR reaction setup: 1X Taq Buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 1µM of each forward and reverse primers (GUS and Hygromycin), 1U of Taq DNA Polymerase and DNA (500 ng). The reaction volume was made up to 25 µl using sterile distilled water.

Chapter 2

b. PCR cycling conditions: 94°C for 30 sec, Annealing temperature (Ta) for 1 min., 72°C for 1fragment, followed by a final extension at 72°C for 10

c. Separation of PCR amplified products by Agarose Gel Electrophoresis

25 µl of PCR amplified products from each reaction along with 2 µl of loading dye (Bromophenol blue) was loaded on 1% agarose gel. The electrophoresis was carried out using 1X TAE buffer (pH-8.0) 1kb or 100separate lane. The PCR bands on the gels were visualized under UV transilluminator and documented.

Table 2.3 List of primers used for analysis of putative transgenic T

2.2.8.1.4 Southern blot analysisGenomic DNA (40 µg) was digested overnight with restriction enzyme EcoR1 and then purified using Qiagen PCR purification kit. The digested DNA was electrophoresed on a 1% agarose gel. The DNA from the gel was transferred to a nylon membrane using the capillary transfer method aswas treated with Depurination solution (0.2(1.5 M NaCl, 0.5 N sodium hydroxide) for 30 min and Neutralizing solution (1M TrisHCl, pH 7.4, 1.5 M NaCl) for 30 capillary transfer in 20X SSC (3

68

Initial denaturation step at 94°C for 5 min, 30 cycles at sec, Annealing temperature (Ta) for 1 min., 72°C for 1

fragment, followed by a final extension at 72°C for 10 min and hold at 4°C. Separation of PCR amplified products by Agarose Gel Electrophoresis

µl of PCR amplified products from each reaction along with 2 µl of loading dye loaded on 1% agarose gel. The electrophoresis was carried out 8.0) 1kb or 100 bp molecular weight marker was run in a

separate lane. The PCR bands on the gels were visualized under UV transilluminator

of primers used for analysis of putative transgenic T0 Plants

.1.4 Southern blot analysis digested overnight with restriction enzyme EcoR1 and then

purified using Qiagen PCR purification kit. The digested DNA was electrophoresed on a 1% agarose gel. The DNA from the gel was transferred to a nylon membrane using the capillary transfer method as described in Sambrook et al., (1989). Briefly, the gel was treated with Depurination solution (0.2 N HCl) for 15 min, Denaturation solution

N sodium hydroxide) for 30 min and Neutralizing solution (1M TrisM NaCl) for 30 min. The DNA was transferred to nylon membrane by

capillary transfer in 20X SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) overnight and

min, 30 cycles at min/ Kb of

min and hold at 4°C. Separation of PCR amplified products by Agarose Gel Electrophoresis

µl of PCR amplified products from each reaction along with 2 µl of loading dye loaded on 1% agarose gel. The electrophoresis was carried out

bp molecular weight marker was run in a separate lane. The PCR bands on the gels were visualized under UV transilluminator

digested overnight with restriction enzyme EcoR1 and then purified using Qiagen PCR purification kit. The digested DNA was electrophoresed on a 1% agarose gel. The DNA from the gel was transferred to a nylon membrane using

(1989). Briefly, the gel N HCl) for 15 min, Denaturation solution

N sodium hydroxide) for 30 min and Neutralizing solution (1M Tris-min. The DNA was transferred to nylon membrane by

M sodium citrate, pH 7.0) overnight and

Chapter 2

69

cross-linked by UV irradiation at 254 nm for 2 min. The membrane was pre-hybridised for 4 h at 45°C and DIG labeled full-length PCR-amplified GUS probe (1029 bp) and hptII probe (355 bp) were prepared by random priming method using the Roche DIG High Prime DNA Labeling and Detection Starter kit II (Catalog no. ). The DIG labeled probes were denatured and added to the fresh pre-hybridization solution for overnight incubation at 50-53°C. The membrane was washed sequentially in 2X SSC+ 0.1% SDS; 0.5X SSC+ 0.1% SDS at 68°C for 15 min each 3 times, then washed with blocking solution and with Anti-Digoxigenin for 30 min each. Finally for detection, detection buffer was used for 5 min and applied CSPD as substrate. The membrane was then wrapped in saran wrap and incubated for 2 h at 37°C. Scanning and recording of images was performed using Phosphorimager like night OWL instrument. 2.3 EXPERIMENTAL RESULTS 2.3A Optimization of genetic transformation of Cenchrus ciliaris using

Agrobacterium tumefaciens Experiments were carried out to optimize Agrobacterium mediated transformation of Cenchrus ciliaris genotype IG-3108. For standardization of genetic transformation protocol using Agrobacterium strain (EHA 105 consisting of pCAMBIA 1301 and 1305.1), shoot apices and calli induced from immature inflorescence were used as explants for agro-infection and transgenic shoots could be developed only through multiple shoot formation induced from shoot apices. 2.3.1 Hygromycin sensitivity test Shoot apex explants were cultured on MS media with different levels of hygromycin (0, 5, 10, 15, 20, 25, 30 and 50 mg/l) to know the sensitivity of shoot regeneration to the antibiotic. All the shoots were not able to survive on MS media with 3 mg/l TDZ and 30 mg/l Hygromycin, only 3% shoots survived at a concentration of 25 mg/l. Above this level shoots completely died. Therefore 30 mg/l Hygromycin was used as optimum concentration for selection of putative transformants (Table 2.4).

Chapter 2

Table 2.4 Hygromycin sensitivity test for shoot apices after exposure to a concentration of 0-50 mg/l hygromycin B.

a-e Data with the same superscript letters are not significantly different from each other at the 5% level based on Duncan’s Multiple Range test.

A Each experiment was replicated at least three times.

2.3.2 Standardization of transformation protocol 2.3.2A Bacterial cell density To detect the most suitable ODtested; 0.6, 0.8, 1.0, 1.2, 1.5, 2.0. Bacterial cell concentration measured by optical density (O.D) of bacterial suspension is directly proportional to their cell number. The optimal OD was determined by observing the blue spotby the Agrobacterium. When using 1301 and 1305.1, the highest number of GUS expressing apices were observed with OD600nm = 1.0. However, the number of apices expressing GUS at ODobserved to be 15 ± 2.9 and 18 ± 3.7 for pCAMBIA 1301 and pCAMBIA 1305.1, respectively. The lowest number of GUS positive shoot apices was recorded at= 2.0 (0.66-1.0) (Table 2.5).

With higher densities of Agrobacterium transformation frequency was observed. was observed following inoculation with high densities of probably due to increased production of toxins to the receptor cehad significant influence on transformation efficiency

70

Table 2.4 Hygromycin sensitivity test for shoot apices after exposure to a concentration

same superscript letters are not significantly different from each other at the 5% level based Each experiment was replicated at least three times.

2.3.2 Standardization of transformation protocol l density

To detect the most suitable OD600nm for Agrobacterium culture, six different levels were tested; 0.6, 0.8, 1.0, 1.2, 1.5, 2.0. Bacterial cell concentration measured by optical density (O.D) of bacterial suspension is directly proportional to their cell number. The optimal OD was determined by observing the blue spots on the shoot apex transformed

. When using Agrobacterium strain EHA 105 with pCAMBIA 1301 and 1305.1, the highest number of GUS expressing apices were observed with

= 1.0. However, the number of apices expressing GUS at OD600n

15 ± 2.9 and 18 ± 3.7 for pCAMBIA 1301 and pCAMBIA 1305.1, number of GUS positive shoot apices was recorded at

Agrobacterium cells (OD600nm > 2.0), a reduction in mean transformation frequency was observed. A decline indicates transformation frequency was observed following inoculation with high densities of Agrobacteriumprobably due to increased production of toxins to the receptor cells. Thus, cell density had significant influence on transformation efficiency.

Table 2.4 Hygromycin sensitivity test for shoot apices after exposure to a concentration

same superscript letters are not significantly different from each other at the 5% level based

, six different levels were tested; 0.6, 0.8, 1.0, 1.2, 1.5, 2.0. Bacterial cell concentration measured by optical density (O.D) of bacterial suspension is directly proportional to their cell number. The

s on the shoot apex transformed EHA 105 with pCAMBIA

1301 and 1305.1, the highest number of GUS expressing apices were observed with 600nm 1.0 was

15 ± 2.9 and 18 ± 3.7 for pCAMBIA 1301 and pCAMBIA 1305.1, number of GUS positive shoot apices was recorded at OD600nm

2.0), a reduction in mean A decline indicates transformation frequency

Agrobacterium cells, lls. Thus, cell density

Table 2.5 Influence of Agrobacteriumtransient GUS expression

* Significant at P= 0.05 A Explants were co-cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 during an infection time of 30

minutes, then co-cultivated for 3 days in coB For each OD600nm studied, experiments were repeated three times with 60 explants.C Transient GUS expression frequency (%) = (Number of explants showing GUS

of explants inoculated) *100a-c Statistical analysis was carried out using

the same letter were not significantly different , as indicated by LSD (P= 2.3.2B Duration of infection and coGUS expression frequency is influenced by the period of exposure to explant with Agrobacterium cells. Tstudied. The explants were incubated for 30 minutes with O.D600 = 1.0 showed highest efficiency of transformation than those incubated for 10 min or 20 min, whereas exposure to decline in transformation frequency (Table 2.6)unfavorably affected the 2.3.2C Co-cultivation period We conducted a transient expression experiment after coconditions (OD600nm = 1.0 and inoculation time of 30 min.), the effect of differing duration of co-cultivation period was studied.hours (light/dark) photoperiod from 0undetectable in cells cofor 2 days, up to the highest for 3 days. Extending the codid not increase GUS activity, but caused necrosis and death of cells (Table 2.7).

71

Agrobacterium concentration (OD 600nm values) on the frequency of transient GUS expression (%) in shoot apex explants of Cenchrus ciliaris

cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 during an infection time of 30 cultivated for 3 days in co-cultivation medium supplemented with 400studied, experiments were repeated three times with 60 explants.

Transient GUS expression frequency (%) = (Number of explants showing GUS-positive spots / Total number of explants inoculated) *100 Statistical analysis was carried out using one-way ANOVA (DMRT). Mean within same columns followed by the same letter were not significantly different , as indicated by LSD (P=0.05)

Duration of infection and co-culture GUS expression frequency is influenced by the period of exposure to explant with

The effect of changing the length of co-cultstudied. The explants were incubated for 30 minutes with Agrobacterium

showed highest efficiency of transformation than those incubated for 10 min or 20 min, whereas exposure to Agrobacterium for over 40 minutes resulted in line in transformation frequency (Table 2.6). Prolonged inoculation time

unfavorably affected the explant owing to overgrowth of Agrobacterium

cultivation period We conducted a transient expression experiment after co-cultivation.

= 1.0 and inoculation time of 30 min.), the effect of differing cultivation period was studied. Co-cultivation was carried out in the 16/8

hours (light/dark) photoperiod from 0-5 days. Transient GUS expression was undetectable in cells co-cultivated for 30 min, but could be observed in cells cofor 2 days, up to the highest for 3 days. Extending the co-cultivation period to 4

activity, but caused necrosis and death of cells (Table 2.7).

Chapter 2

values) on the frequency of Cenchrus ciliaris A

cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 during an infection time of 30

cultivation medium supplemented with 400 µM acetosyringone.

positive spots / Total number way ANOVA (DMRT). Mean within same columns followed by

GUS expression frequency is influenced by the period of exposure to explant with culturing period was

Agrobacterium cells at showed highest efficiency of transformation than those incubated for 10

for over 40 minutes resulted in . Prolonged inoculation time Agrobacterium on explants.

cultivation. Using optimum = 1.0 and inoculation time of 30 min.), the effect of differing the

cultivation was carried out in the 16/8 Transient GUS expression was

cultivated for 30 min, but could be observed in cells co-cultivated cultivation period to 4-5 days

activity, but caused necrosis and death of cells (Table 2.7).

Chapter 2

Table 2.6 Influence of duration of cofrequency of transient GUS expression (%) in shoot apex explants of

* Significant at P= 0.05 A Explants were co-cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 at OD

cultivated for 3 days in co-cultivation medium containing 400B For each duration studied, experiments were repeated three times with 60 explants.C Transient GUS expression frequency (%) = (number of explants showing

of explants inoculated) *100 a-d Statistical analysis was carried out using one

the same letter were not significantly different Table 2.7 Influence of duration of coexpression (%) in shoot apex explant of

* Significant at P= 0.05 A Explants were co-cultured with EHA 105 harbouring pCAMBIA 1301 or

time of 30 minutes in co-cultivation medium supplemented with 400B For each duration period (day) studied, experiments were repeated three times with 60 explants.C Transient GUS expression frequency (%) = (number of expl

of explants inoculated) *100 a-d Statistical analysis was carried out using one

the same letter were not significantly dif

72

Table 2.6 Influence of duration of co-culture on Agrobacterium inoculums on frequency of transient GUS expression (%) in shoot apex explants of Cenchrus ciliaris

cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 at OD600nm

cultivation medium containing 400 µM acetosyringone. For each duration studied, experiments were repeated three times with 60 explants. Transient GUS expression frequency (%) = (number of explants showing GUS-positive spots / total number

Statistical analysis was carried out using one-way ANOVA (DMRT). Mean within same columns followed ere not significantly different, as indicated by LSD (P=0.05)

Table 2.7 Influence of duration of co-cultivation period on the frequency of transient GUS expression (%) in shoot apex explant of Cenchrus ciliaris A

cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 at OD600nm 1.0 and an infection cultivation medium supplemented with 400 µM acetosyringone. (day) studied, experiments were repeated three times with 60 explants.frequency (%) = (number of explants showing GUS-positive spots

Statistical analysis was carried out using one-way ANOVA (DMRT). Mean within same columns followed by ere not significantly different, as indicated by LSD (P=0.05)

inoculums on the Cenchrus ciliaris A

600nm 1.0 then co-

positive spots / total number

n within same columns followed by

cultivation period on the frequency of transient GUS

1.0 and an infection

(day) studied, experiments were repeated three times with 60 explants. positive spots/ total number

way ANOVA (DMRT). Mean within same columns followed by

Chapter 2

73

2.3.2D Acetosyringone Plant specific phenolic compounds that stimulate the expression of vir genes of Agrobacterium are essential for gene transfer. In monocots plants, where these compounds aren’t synthesized, adding up of phenolic compounds such as acetosyringone throughout plant / bacterial interaction supports the gene transfer. Using the optimal conditions (OD600nm = 1.0, inoculation period of 30 min and co-cultivation period of 3 days), the effect of varying concentration of acetosyringone (0-500 µM) in co-cultivation medium was studied. GUS expression was not found when acetosyringone was not added into the co-cultivation medium. It was observed that increasing the concentration of acetosyringone from 100 µM to 400 µM increase the GUS expression efficiency, the maximum frequency of 32.3 ± 1.4 for pCAMBIA 1305.1 was obtained on medium containing 400 µM acetosyringone (Table 2.8). These results suggested that addition of acetosyringone in co-cultivation medium significantly influenced GUS expression frequency. It is crucial for successful transformation of Cenchrus ciliaris. 2.3.2E Infection under negative pressure Vacuum infiltration is one of the simplest methods of Agrobacterium mediated transformation. Vacuum treatment to infiltrate tissues with Agrobacterium has been successfully used in transformation of cereals and grasses. In this experiment, a negative pressure of 0.5 x 105 Pa was produced through vacuum pump in a vacuum desiccator, which resulted in increased GUS expression frequency in contrast to that found at atmospheric pressure. It has been suggested that vacuum pump produced a negative pressure surroundings that consequences in an increase in effective Agrobacterium volatilization, a provision beneficial to the transfer of an alien gene into plant cells. When negative pressure was given to shoot apices immersed in Agrobacterium cells for more than 30 min. resulted in explants getting entirely colonized by Agrobacterium, making it more difficult to remove in the pre-regeneration medium, finally resulting in loss of shoot apices and their consequent growth. In the current study, when combined with standardized protocol of vacuum infiltration assisted transformation of shoot apices of Cenchrus genotype there was an improvement in the transformation efficiency. The highest transformation percentage (17.3 ± 1.4a) was obtained with a negative pressure of 0.5 x 105 Pa compared to 13.6 ± 1.3a without the pressure (Table 2.9).

Chapter 2

Table 2.8 Influence of acetosyringone concentration in the cofrequency of transient GUS expression (%) in shoot apex explant of

* Significant at P= 0.05 A Explants were co-cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1

time of 30 minutes In co-cultivated for 3 days.B For each concentration studied, experiments were repeated three times with 60 explants.C Transient GUS expression frequency (%) = (number of explants showing GUS

of explants inoculated) *100 a-e Statistical analysis was carried out using one

the same letter were not significantly different, as indicated by LSD (P= Table 2.9 Influence of vaccum infiltration treatment on the frequency of transient GUS expression (%) in shoot apex explant of

A Explants were co-cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 at ODtime of 30 minutes and co-cultivated for 3 days on coacetosyringone.

B For each concentration studied, experiments were repeated three times with 60 explants.C Transient GUS expression frequency (%) = (number of explants showing GUS

of explants inoculated) *100 a Statistical analysis was carried out using one

the same letter were not significantly diff 2.3.3 Regeneration of putative transformed plantsResults from 10 independent experiments are summarized inpCAMBIA 1301 (Figure 2.5)procedure, 90 shoot apices (Figure 2.5awere immersed in Agrobacterium

74

cetosyringone concentration in the co-cultivation medium on the frequency of transient GUS expression (%) in shoot apex explant of Cenchrus ciliaris

cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 at OD600nm 1.0 and an infection cultivated for 3 days.

For each concentration studied, experiments were repeated three times with 60 explants. Transient GUS expression frequency (%) = (number of explants showing GUS-positive spots / total number Statistical analysis was carried out using one-way ANOVA (DMRT). Mean within same columns followed by the same letter were not significantly different, as indicated by LSD (P=0.05)

Table 2.9 Influence of vaccum infiltration treatment on the frequency of transient GUS expression (%) in shoot apex explant of Cenchrus ciliaris A

cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 at OD600nm 1.0 and an infection cultivated for 3 days on co-cultivation medium containing 400

For each concentration studied, experiments were repeated three times with 60 explants. frequency (%) = (number of explants showing GUS-positive spots / total number

Statistical analysis was carried out using one-way ANOVA (DMRT). Mean within same columns followed by ere not significantly different, as indicated by LSD (P=0.05)

2.3.3 Regeneration of putative transformed plants Results from 10 independent experiments are summarized in Table 2.10

(Figure 2.5) and 1305.1 (Figure 2.6). Using optimal transformation (Figure 2.5a-b, 2.6a-b) for each plasmid (Figure 2.5c, 2.6c)

Agrobacterium suspension [MS inf- medium (before co-

cultivation medium on the Cenchrus ciliarisA

1.0 and an infection

spots / total number way ANOVA (DMRT). Mean within same columns followed by

Table 2.9 Influence of vaccum infiltration treatment on the frequency of transient GUS

1.0 and an infection cultivation medium containing 400 µM

positive spots / total number way ANOVA (DMRT). Mean within same columns followed by

Table 2.10-11 for . Using optimal transformation

(Figure 2.5c, 2.6c) -culturing in

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this medium, grown Agrobacterium culture was centrifuged at 6000 rpm at 25ºC for 10min. and then re-suspended in MS-inf medium)] with OD600 1.0 for 30 min under vacuum condition (Figure 2.5d, 2.6d). Excess bacterial suspension was removed from the explants by placing them on sterile Whatman filter paper and after that shoot apices were co-cultivated for 3 days on co-cultivation medium supplemented with 400 µM acetosyringone. Where the shoot apices were subcultured on the first selection medium directly after co-cultivation, these shoot apices sufferd from the stress of Agrobacterium and selection medium. To avoid this problem, after co cultivation, shoot apices were subcultured to recovery medium. In this medium, shoots were multiplied without selection agent containing cefotaxime to remove the Agrobacterium growth. After that, shoot apices were rinsed with cefotaxime (250 mg/l) for 10 minutes, subsequently washed thoroughly with sterile double distilled water for 2-3 times, blotted on filter paper and subcultured onto recovery medium (Figure 2.5f, 2.6f) for one week. Later than recovery phase, shoot apices were transferred to first selection medium (Figure 2.5g, 2.6g) (Table 2.2) containing 30 mg/l hygromycin for 15 days and after that again subcultured on II selection medium (Figure 2.5h, 2.6h) for 15 days to allow the better multiplication of transformed shoots. Shoot apices remained alive after selection, were transferred to pre-regeneration medium (Figure 2.5i-j, 2.6i-j) for 3-5 weeks to stimulate the multiplication of transgenic shoots from transformed shoot apices. 14 transformed plants showed GUS expression in leaves with pCAMBIA 1301 and 17 with pCAMBIA 1305.1. These transformed shoots were then multiplied on regeneration medium (Table 2.10-11) for 2-3 weeks. The well grown elongated shoots with so many leaves were rooted on rooting medium (Figure 2.5k, 2.6k) and then hardened in sterile pots on sterilized vermiculite and solarite. And then covered with polythene bags to retain moisture/ humidity and nutrients were given through Hoagland's solution (Figure 2.5l-m, 2.6l-m).

Overall results from 10 independent experiments are summarized in Table 2.10, a total of 706 explants were cocultivated with Agrobacterium and 47 transferred to II selection medium, 14 survived from selection and 6.34 % ± 0.86 multiplied on regeneration

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medium. Stable transformation efficiency in these experiments averaged for pCAMBIA 1301 was 1.42 ± 0.34 and ranged between 1.3 to 2.8 %.

Similarly 699 shoot apices were1305.1 and 46 plants were traselection. And 6.97 % ± 1.2 plants transformation efficiency of 1.37% ± 0.54 Table 2.10 Summary of Agrobacteriumapex explants of Cenchrus ciliaris

A Shoot apex were co-cultured with EHA minutes and co-cultivated for 3 days on co

Table 2.11 Summary of Agrobacteriumapex explants of Cenchrus ciliaris

A Shoot apex explants were co-cultured with EHA 105 harbouring pCAMBIA 1305.1 at ODinfection time of 30 minutes and co400µM acetosyringone.

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medium. Stable transformation efficiency in these experiments averaged for pCAMBIA ranged between 1.3 to 2.8 %.

were co-cultured with Agrobacterium containing pCAMBIA transferred to II selection medium and 17 survived the plants regenerated on regeneration medium. We obtained a

transformation efficiency of 1.37% ± 0.54 and ranged between 1.2 to 4.3% (Table 2.11).

Agrobacterium- mediated transformation of 3 days old shoot Cenchrus ciliaris cv. 3108

cultured with EHA 105 harbouring pCAMBIA 1301 at OD600nm 1.0 and an infection time of 30 cultivated for 3 days on co-cultivation medium supplemented with 400µM acetosyringone.

Agrobacterium- mediated transformation of 3 days old shoot Cenchrus ciliaris cv. 3108

cultured with EHA 105 harbouring pCAMBIA 1305.1 at OD600nminfection time of 30 minutes and co-cultivated for 3 days on co-cultivation medium supplemented with

medium. Stable transformation efficiency in these experiments averaged for pCAMBIA

containing pCAMBIA 17 survived the We obtained a

and ranged between 1.2 to 4.3% (Table 2.11).

mediated transformation of 3 days old shoot

1.0 and an infection time of 30 cultivation medium supplemented with 400µM acetosyringone.

mediated transformation of 3 days old shoot

600nm 1.0 and an cultivation medium supplemented with

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Figure 2.5 Agrobacterium-mediated transformation and plant regeneration in Cenchrus ciliaris using strain EHA105 harboring pCAMBIA 1301: (a) Seeds grown on MS medium containing 1 mg/l TDZ; (b) Three day old shoot apices grown on pre-culture medium; (c) Culture of Agrobacterium strain EHA 105 harboring pCAMBIA 1301; (d) Co-culturing Agrobacterium with shoot apices on MS liquid medium, (e) No GUS expression in leaves of untransformed control shoot apices and transient GUS expression (visible as blue spots) in leaves of transformed shoots; (f) Seven day old plants on resting medium; (g) Shoots growing on hygromycin B supplemented Ist selection medium; (h) Transformed plants after IInd selection medium; (i) Transformed plants growing on regeneration medium; (j) Shoot elongation and multiplication, (k-m) Rooting and hardening of transformed plants in green house.

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Figure 2.6 Agrobacterium-mediated transformation and plant regeneration in Cenchrus ciliaris using strain EHA 105 harboring pCAMBIA 1305.1: (a) Seeds grown on MS medium supplemented with 1 mg/l TDZ, (b) Three day old shoot apices grown on pre-culture medium; (c) Culture of Agrobacterium strain EHA 105 harboring pCAMBIA 1305.1; (d) Co-culturing Agrobacterium with shoot apices on MS liquid medium; (e) Untransformed control leaves and GUS expression in transformed leaves; (f) Seven day old plants on resting medium; (g) Shoots growing on hygromycin B supplemented Ist selection medium, (h) Transformed plants after IInd selection medium; (i) Transgenic (T0) plant growing on regeneration medium; (j) Shoot elongation and multiplication, (k-m) rooting and hardening of transformed plants in green house.

2.3.4 Agrobacteriumembryogenic calli

Callus induced from immature inflorescence explants of genotype IGmedium with 3 mg/l 2,used for Agrobacteriumcontaining construct pCAMBIA 1301 and 1305.1 calli (Figure 2.7a, 2.8aafter that the GUS expression efficiencycultured in ten different experiments and the mean ± 2.3 and 20.5% ± 1.7vectors were used, respectivelyon recovery medium for one week and then transferred on the selection medium2.5d-e, 2.6b). Only 5.96 % ± 0.78 and 9.02 % ± 1.5on selection medium for pCAMBIA 1301 and pCAMBIA 1305.1 respectively,transformed calli could not be regenerated onwith 0.25 mg/l 2,4-D) (Table

Table 2.12 GUS activity of calli of Immature Inflorescence explants of innoculated with Agrobacterium tumefaciens

A calli were co-cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 at ODof 30 minutes and co-acetosyringone.

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Agrobacterium–mediated transient GUS gene expression in embryogenic calli

Callus induced from immature inflorescence explants of genotype IGmg/l 2,-4D supplemented with 0.5mg/l BA (describe on chapter 1) was

Agrobacterium- mediated transformation. Agrobacteriumcontaining construct pCAMBIA 1301 and 1305.1 were co-cultured

a). The co cultivation period was for 3 days at 28ºC in darkafter that the GUS expression efficiency was analysed. A total of

erent experiments and the mean GUS expression efficiency of 24.8 %± 2.3 and 20.5% ± 1.7 were observed when pCAMBIA 1305.1 and pCAMBIA 1301 vectors were used, respectively (Table 2.12). After co-cultivation calli were subcultured on recovery medium for one week and then transferred on the selection medium

5.96 % ± 0.78 and 9.02 % ± 1.5 of the calli survived and multiplied on selection medium for pCAMBIA 1301 and pCAMBIA 1305.1 respectively,transformed calli could not be regenerated on the regeneration medium (

D) (Table 2.13-14).

Table 2.12 GUS activity of calli of Immature Inflorescence explants of Agrobacterium tumefaciens EHA105.

cultured with EHA 105 harbouring pCAMBIA 1301 or 1305.1 at OD600nm -cultivated for 3 days on co-cultivation medium supplemented with 400

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mediated transient GUS gene expression in

Callus induced from immature inflorescence explants of genotype IG-3108 on MS 4D supplemented with 0.5mg/l BA (describe on chapter 1) was

Agrobacterium strain EHA 105 cultured with embryogenic

for 3 days at 28ºC in dark and 600 calli, were co-efficiency of 24.8 %

were observed when pCAMBIA 1305.1 and pCAMBIA 1301 cultivation calli were subcultured

on recovery medium for one week and then transferred on the selection medium (Figure of the calli survived and multiplied

on selection medium for pCAMBIA 1301 and pCAMBIA 1305.1 respectively, but the regeneration medium (MS+3 mg/l BA

Table 2.12 GUS activity of calli of Immature Inflorescence explants of Cenchrus ciliaris

1.0 and an infection time cultivation medium supplemented with 400 µM

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Table 2.13 Summary of Agrobacteriumfrom immature inflorescence explant of

A Calli were co-cultured with EHA 105 harbouring pCAMBIA 1301 at ODminutes and co-cultivated for 3 days on co

Table 2.14 Summary of Agrobacteriumfrom immature inflorescence explant of

A Calli were co-cultured with EHA 105 harbouring pCAMBIA 1305.1 at ODminutes and co-cultivated for 3 days on co

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Agrobacterium- mediated transformation of two monthnflorescence explant of Cenchrus ciliaris cv.3108

cultured with EHA 105 harbouring pCAMBIA 1301 at OD600nm 1.0 and an infection time of 30 cultivated for 3 days on co-cultivation medium containing 400 µM acetosyringone.

Agrobacterium- mediated transformation of two months old calli nflorescence explant of Cenchrus ciliaris cv.3108

cultured with EHA 105 harbouring pCAMBIA 1305.1 at OD600nm 1.0 and an infection time of 30 cultivated for 3 days on co-cultivation medium containing 400 µM acetosyringone.

mediated transformation of two months old calli

1.0 and an infection time of 30 µM acetosyringone.

mediated transformation of two months old calli

1.0 and an infection time of 30 µM acetosyringone.

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Figure 2.7 Agrobacterium-mediated transformation of embryogenic calli of Cenchrus cilliaris using binary vector pCAMBIA 1301: (a) Embryogenic calli co-cultured with Agrobacterium on MS-inf liquid medium; (b-c) Transient GUS expression in calli- untransformed control callus and transformed callus with blue spot; (d-e) Transformed calli on selection medium.

Figure 2.8 Agrobacterium-mediated transformation of embryogenic calli of Cenchrus cilliaris using binary vector pCAMBIA 1305.1: (a) Embryogenic calli co-cultured with Agrobacterium on MS- inf liquid medium; (b-c) Transient GUS expression in calli- untransformed control callus and transformed callus with blue spot; (d-e.) Transformed calli on selection medium.

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2.3.5 Histochemical GUS assay Histochemical staining revealed transient GUS expression in shoot apex or calli explants after 3 days of co-cultivation with pCAMBIA 1301 and pCAMBIA 1305.1 (Figure 2.5-2.8). Strong GUS expression was detected in shoot apices (Figure 2.5e-2.6e) and calli explants (Figure 2.7b-c, 2.8c-d). Leaves of putative transgenic T0 plants were stained positive for GUS activity, while leaf samples from non-transformed plantlets were not blue (Figure 2.5e-2.7c, 2.8d).

2.3.6 Molecular analysis of putative transgenics (T0) Genomic DNA was extracted from the leaves of non-transformed control plant, 14 and 17 transformed plants obtained after selection and regeneration for pCAMBIA 1301 and pCAMBIA 1305.1, respectively (Figure 2.9a, 2.10a). 2.3.6.1 PCR analysis To confirm the presence of transgenes in putative transformants, polymerase chain reaction (PCR) was performed using nptII and hptII genes using extracted genomic DNA. Expected GUS PCR product (1029 bp for pCAMBIA1301 and 406 bp for pCAMBIA 1305.1) could be amplified by using genomic DNA from 14 and 17 transformant T0 lines tested while no amplified product was observed in non-transformed control plant (Figure 2.9b, 2.10b). Out of 31 GUS positive transgenic plants, 14 (pCAMBIA 1301) and 17 (pCAMBIA 1305.1) T0 plants were again tested by using hptII gene specific primers and showed amplification of 694 bp and 355 bp respectively (Figure 2.9c, 2.10c). 2.3.6.2 Southern blot analysis (a) Southern blot analysis of transgenic plants containing pCAMBIA 1301

vector Stable integration of the transgene in PCR positive plants was confirmed by Southern analysis. Genomic DNA was digested with the help of restriction enzyme EcoR1 and hybridized to a 1029 bp probe specific to GUS gene. Out of 14 plants identified through PCR, 11 plants showed the GUS transgene, thus confirming their stable transgenic

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status. In one of hybridizations, 6 transgenic lines (T01, T02, T04, T05, T07, T08) contained a single copy of gene. However, 5 transgenic lines (T09, T010, T012, T013, T014) carried 2 to 3 copies of gene (Figure 2.9d). Southern hybridization analysis confirmed the integration of the T-DNA region into the transformed plant genome.

Figure 2.9 Molecular analyses of transgenic plants of Cenchrus ciliaris through pCAMBIA 1301 (a) Isolation of genomic DNA of putative transgenic plants; Lane1: Marker (M-BDNA/hindIII

ladder); Lane2-15: Putative transgenic plants (T01 - T014). (b) PCR amplification for detection of 1029 bp of the GUS gene; Lanes1: Marker (M-1Kb, 100bp

ladder); Lanes2-14, 16: Putaitive gusA transgenic plants (T01 - T014) Lane 15: control. (c) 694bp of the hptII gene in transgenic plants. Lanes1: M (1Kb ladder), Lane11: Control

(Untransformed plant); Lane 2-10, 12-16: Putative transgenic plants (T01 - T014). (d) Southern blot analysis of transgenic plants. Lanes1-6, 8-12: transgenic plants (T01 - T014)

Lane7: control (untransformed plant). Genomic DNA was digested with EcoR1 and hybridized with GUS probe (1029bp).

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(b) Southern blot analysis of transgenic plants containing pCAMBIA 1305.1 vector

Genomic DNA was digested with enzyme EcoRI and 355 bp probe for hptII gene coding region was used. No hybridization signals could be detected for the negative control (non-transformed plant). Out of 17 plants identified through PCR, 10 plants showed the hptII transgene, thus confirmed the integration of the T-DNA region into the transformed plant genome. Among the 10 lines tested, all lines (T05, T07, T09, T010, T011, T013, T014, T015, T016, T017) showed the presence of 2 to 3 copies of the transgene (Figure 2.10d), which, together with the results of the histochemical GUS analysis, indicate that the hptII gene was integrated into the plant genome.

Figure 2.10 Molecular analyses of transgenic plants of Cenchrus ciliaris through pCAMBIA 1305.1 (a) Isolation of genomic DNA of putative transgenic plants; Lane1: Marker (M-BDNA/ hindIII

ladder); Lane2-18: Putative transgenic plants (T01 - T017). (b) PCR amplification for detection of 406 bp of the GUS gene; Lane1: Control (untransformed plant);

Lane12: M (1Kb ladder); Lanes: 2-11, 13-19: Putaitive gusA transgenic plants (T01 - T017). (c) 355 bp of the hptII gene in transgenic plants. Lane1: M (1Kb ladder), Lane2, 4-19: Putative

transgenic plants (T01 - T017) Lane 3: Control. (d) Southern blot analysis of transgenic plants. Lanes: 1-6, 8-10: transgenic plants (T05 - T017)

Lane7: control (untransformed plant). Genomic DNA was digested with EcoR1 and hybridized with hptII probe (355bp).

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2.4 DISCUSSION Traditional plant breeding approaches towards genetic improvement of any crop depends on reproductive compatibility between two individuals which are used as donar and recipient parents during introgressive hybridization. But, when desirable genes have to be transferred across reproductive barriers, recent techniques of in vitro genetic manipulation could be used.

With the introduction of molecular biology and genetic engineering methods it is now possible to overcome the difficulties linked with conventional plant propagation. Genetic transformation of crop plants using Agrobacterium helps to make specific changes in the genome by the addition of one or only some genes. However, reliable and efficient transformation method is a pre-requisite to achieve this goal. Development of efficient transformation system with Agrobacterium has been a main limitation in many grasses including buffel grass (Cenchrus ciliaris). Cenhrus is an apomictic grass species, its genetic improvement through conventional breeding methods is difficult, time consuming and presently restricted to the selection of elite lines from natural variants. The clones approach in apomictic species imposes a restriction on the genetic variability within a variety. A reduced genetic basis of any widely used crop represents a threat, giving pests and pathogens an opportunity of becoming virulent with a single mutation. Once having become virulent, the pathogen is capable of attacking all plants since all are equally susceptible. As expected, such an event has occurred in the form of an epidemic of buffel grass blight caused by Magnaporthe grisea (Rodriguez et al., 1999). On the other hand, apomixis may facilitate varietal improvement by genetic transformation, since no further breeding is required to fix the transferred character (Vielle Calzada et al., 1996). Hence, genetic transformation would be a powerful tool for improvement of this pasture grass. An efficient protocol for genetic transformation of this grass, for example genetic manipulation of lignification to increase forage digestibility, would be very useful (Cherney et al., 1991).

Preliminary studies of transient expression of a reporter gene following particle bombardment have been reported in buffel grass (Ross et al., 1995; Bhat et al., 2001) and are limited to biolistic methods. While microprojectile bombardment has revolutionized the way of genetic transformation of cereals and grasses, there are

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substantial differences seen in the stability, integration and expression of the introduced transgene (Kohli et al., 1999). consequently, there is a necessity for alternate way such as Agrobacterium- mediated transformation, which appears more effective at regenerating transgenic plants with low copy number of transgene. These transgenic plants are more stable in many generations and have shown reduction in gene silencing (Barkat et al., 1997; Shou et al., 2004).

The two most vital steps required for genetic transformation of plants are transfer of foreign DNA into the plant cells and regeneration of plants from transformed cells (Yookongkaew et al., 2007). In several species, transgenic plant recovery is tricky, as the result of cells accessible for gene transfer might not be appropriate for plant regeneration (Komari et al., 1998). Therefore, development of an effortless and valuable approach for gene transfer is of main interest. Within the current study, an efficient protocol of direct and indirect plant regeneration through shoot organogenesis from shoot apices and somatic embryogenesis from explants such as seeds, shoot apices and immature inflorescences had been developed for buffel grass. Somaclonal variations and genetic mutations were low in plants regenerated from shoot apices and apical meristematic cells as compared to in vitro plants regenerated from explants as embryogenic calli (Bregitzer et al., 2002). One possible reason for this low somaclonal variation is also the absence of tissue dedifferentiation steps that are common within the induced callus and initiation of somatic embryo (Hirochika, 1993). Hence, direct shoot induction could be the popular methodology for Agrobacterium- mediated transformation protocol so as to reduce somaclonal variation and genotype dependency found throughout callus-mediated plant regeneration method. Transformation of shoot apex was first proposed in 1988 (McCabe et al., 1988). Cultures of shoot apex may be combined with either Agrobacterium -mediated transformation or particle bombardment (Gould & Magallanes-Cedeno, 1998; Zapata et al., 1999; Goldman et al., 2003; Cho et al., 2003; Yookongkaew et al., 2007).

Two strategies are typically used to develop transgenic plants by transfer of foreign DNA into the shoot apical meristem. One is that transgenic young shoots directly developed from the meristematic cells followed by growth of a partly transgenic reproductive organ. Putative transformants produced by this method will constantly be chimeric. Further

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method is initially, transgenic apical meristem cells are multiplied using given growth regulators (TDZ/ BA/ Kinetin), which might be reprogrammed into the growing stage under in vitro environment (Zhong et al., 1996). Therefore, genetic manipulation in transgenic meristem cells through supplemented with growth regulators to induce multiple shoot formation from shoot apical meristem could regenerate into more sure transformants (Yookongkaew et al., 2007). Among these methods, the second pathway method (Multiple shoot induction) was adopted for transformation protocol in our study.

The transfer process of T-DNA into plant genome is influenced by numerous factors (Cheng et al., 2004; Jones et al., 2005). Standardization of these factors is essential for the establishment of successful Agrobacterium transformation system in monocot plants. While in Cenchrus ciliaris, there has been no report of Agrobacterium-mediated transformation, it was vital to study the effects of the numerous factors on T-DNA delivery. Several factors affecting gene transfer were determined by assaying the activity of reporter gene GUS in leaves of putatively transformed plants after second selection. Few reports of use of shoot apex as explants are available such as in maize (Sairam et al., 2003) and rice (Park et al., 1996; Arockiasamy & Ignacimuthu, 2007; Yookongkaew et al., 2007). Cenchrus seed germinated on 1 mg/l TDZ rather than only MS medium because of, its broke the dormancy period and gave the syncrynous germinanaion (Gurel et al., 2003; Chengalrayan et al., 2005; Chengalrayan and Gallo-megher 2001; Sumlu et al., 2010,). 2.4.1 Optimization of genetic transformation Plant genetic transformation mediated by Agrobacterium tumefaciens has become the most frequently used technique for introduction of foreign genes into plant cells and subsequent regeneration of transgenic plants. Different factors such as bacterial cell density, duration of co-culture and co-cultivation period, use of acetosyringone and effect of vacuum infiltration were tried for optimization of Agrobacterium-mediated transformation of Cenchrus ciliaris. For this standardization using Agrobacterium strain EHA 105 containing construct pCAMBIA 1301 and 1305.1, shoot apices of genotype IG-3108 were used as explants material for Agro- infection and transgenic shoots were multiplied through direct shoot oragnogenesis.

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Hygromycin phosphotransferase (hptII) gene is used as a selectable marker, which confers resistance to the aminoglycoside antibiotic hygromycin. Hygromycin allows understandable discrimination between transgenic and non transgenic tissues. Therefore, plasmid construct containing hygromycin resistance gene in the T-DNA region was used for transformation. Prior to starting the gene transfer experiment it was found that 30 mg/l hygromycin in the selection medium was effective as all the shoots of Cenchrus were died this level. Padmanabhan & Sahi, (2009) also used hygromycin at 25 mg/l for selection of putative transformants in Sesbania drummondii. However Li et al., (2011) found hygromycin at 20 mg/l to be optimum in Saussurea involucrata.

Among the different OD of Agrobacterium tried (0.6, 0.8, 1.0, 1.2, 1.5 and 2.0) (from both the construct), Agrobacterium cell density at 1.0 O.D. was found optimum for transformation. Shoot apices explant co-cultivated with higher O.D. (beyond 1.0) had negative effect on the survival rate of transformed plant cells. Further, death of explants was commonly observed at higher O.D. values along with lower frequency of shoots and shoot buds, possibly due to increased production of toxins to the receptor cells (Gu et al., 2008). Similar responses have been reported earlier (Kumar et al., (2005); Yookongkaew et al., (2007) in rice, Shrawat et al., (2007) in barley. Several workers reported the optimum cell density for agro-infection in O.D. less than 1.0 (Zhao et al., 2000 in sorghum, Kumria et al., 2001 in rice, Sarker & Biswas, 2002 in wheat and Gasparis et al., 2008 in oat. Optimization of the Agrobacterium cell density is essential because at high O.D. levels, the plant tissues are almost wholly colonized via the bacteria, removal of which becomes very challenging throughout the co-cultivation, recovery, selection and consequently regeneration steps. Generally this is accomplished by using antibiotics like cefotaxime at higher level which, in itself, has damaging effects on plant growth.

After optimizating OD value at 1.0, shoot apices incubated for 30 min. with Agrobacterium cells showed significant increase in the efficiency of transformation and GUS expression than those transformed for duration of 10 min or 20 min, while exposure to Agrobacterium for more than 45 minutes resulted in reduced transformation efficiency. Prolonged co-culture period negatively affected the plant tissues due to overgrowth of Agrobacterium cells. Different co-culture periods were observed by

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several reporters Zhao et al., (2000), Howe et al., (2006) in Sorghum and Ishida et al., (2007) in Zea mays, reported 5 min. of co-culture duration as optimum, whereas Sarker & Biswas, (2002) reported 50 min. as ideal for Oryza. Our results are in accordance with Lee et al., (2006) in Dactylis glomerata, wherever they suggested 30 min of co-culture period.

Using the conditions for transformation as OD600nm 1.0 and co-culturing time of 30 minutes, the effect of variable length of co-cultivation period was studied. Co-cultivation period for 2-3 days is mostly thought of on appropriate for Agrobacterium mediated transformation as reported for several plant genus like Trirticum (Amoah et al., 2001; Mitic et al., 2004), Oryza (Forkan, et al., 2004), Panicum (Someleva et al., 2002) and Dichanthium (Kumar et al., 2005). In 2006, Lee and co workers conducted the co-cultivation from 1 to 7 days on Dactylis glomerata calli and observed co-cultivation period of 3 days as optimum. Co-cultivation was carried out from 0-5 days in the photoperiod 16/8 hours (light/dark). Our results showed that GUS expression efficiency increased from 1 to 3 days. Though co-cultivation for over 2 days resulted in slight Agrobacterium growth around the plant tissue, GUS expression was additional compared to shoot apices subcultured for 2 days in transient expression as reporter gene studies. Treatment of co-cultivation period for more than 3 days resulted in over proliferation of Agrobacterium, tissue necrosis and later cell death. Our results are in accordance with the observation of Jacob and Veluthambi, (2003); Wu et al., (2003) and Sharawat et al., (2007), where they observed 3 days co-cultivation period as optimum in barley calli.

Acetosyringone, a phenolic compound is well-known to enhance the virulence of Agrobacterium cells, to facilitate the T-DNA integration and also increase the transformation proportion (Stachel et al., 1985). In monocot plants, where such type of compounds are not naturally synthesized, addition of phenolic compounds such as acetosyringone during plant-bacterial interface supports the gene transfer (Hiei et al., 1994; Nadolska-Orezyk and Orezyk, 2000; Koichi et al., 2002). In the current study with shoot apices explants, 400 µM of acetosyringone gave maximum putative transformants. GUS expression was not seen once acetosyringone was excluded from the co-cultivation medium. It was observed that raising the acetosyringone concentration

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from 100 µM to 400 µM notably increased the GUS efficiency. This study supports the previous reports in a range of species concluding that supplementing acetosyringone throughout co-cultivation increased the quantity of transformant cells within the target tissue in Oryza (Hiei et al., 1994), Triticum (Wu et al., 2003) and Hordeum (Shrawat et al., 2007). Though it has not been reported to be important for Agrobacterium transformation of Hordeum (Tingay et al., 1997; Fang et al., 2002), this phenolic compound has been reported to be a key constituent to successful transformation of rice (Hiei et al., 1997), maize (Ishida et al., 1996) and wheat (Cheng et al., 2003; Wu et al., 2003). The variation in the necessity of acetosyringone for successful plant transformation of cereals may be appropriate differences in the bacterial cell density and co-cultivation period and also within the competency of target tissues (Shrawat et al., 2007).

Vacuum infiltration is one of the simplest methods to improve penetrance of Agrobacterium in different parts of the tissue, and it’s successfully used in transformation of cereals (Dong et al., 2001; Amoah et al., 2001). In our system, a negative pressure of 0.5 x 105 Pa, was used that resulted in an enhancement in effectual Agrobacterium volatilization, a condition beneficial to the transfer of an alien gene into plant tissues (Gu et al., 2008). Negative pressure treatment of shoot apices with Agrobacterium for more than 30 min resulted in explant tissues being completely colonized by the Agrobacterium. Our results are similar to with Shrawat et al., (2007) who reported that vacuum infiltration increased the transformation rates in Hordeum.

In our study, the shoot apices immersed in Agrobacterium suspension at an OD600nm 1.0 for 30 min under vacuum treatment followed by co-cultivation for 3 days on co-cultivation medium gave the highest transformation efficiency. This optimized protocol could be used for transferring useful genes to Cenchrus for its genetic improvement.