**3.1 Biological way to introduce DNA into plant cell**

Nature has offered *Agrobacterium* the ability to transfer some part of DNA from plasmid to plant cell. This T-DNA (transfer DNA) naturally causes callus formation on plant's parts termed as crown gall disease. However, this is multifarious procedure that involves two biological systems; bacteria and plant cell and success is subjected to compatibility. Unsurprisingly virulence story of *Agrobacterium* is the key for tumor induction. This virulence provokes by simple carbohydrates and phenolic compounds that are released by injured plant tissue. After this initiative, vir genes activate and produce proteins. These proteins hold the charge of transformation that include scratch of T-DNA, carry, direct towards the plant cell and finally integrate into plant genome. Naturally this T-DNA contains genes that are involved in biosynthesis of plant hormones that are involved in uncontrolled proliferation of plant cell leading to callus formation.

Soybean: Plant Manipulation to *Agrobacterium* Mediated Transformation 303

glyphosate (Hinchee et al., 1988). Modification to regeneration protocol is essential to get high level of transformants. Greater number of mitotic cycles are required before embryo initiation and production of plants with transformed germ lines cells. EHA101 was found more potent to transform soybean immature cotyledons and recovery of transformed plants over LBA4404 (Parrott et al., 1989). However, McKenzie & Cress (1992) were able to get transformed plants from cotyledon and hypocotyl explants from 10 days old seedlings working with LBA4404 harboring pBI121. Transformation efficiency is not dependent only on Agrobacterium genotype but soybean cultivar, age of explant and other conditions also influence. Trick & Finer (1997) introduced sonication assisted *Agrobacterium* mediated transformation (SAAT) system. SAAT permits efficient delivery of T-DNA to large number of plant cell in a variety of different plant tissues. In soybean, GUS expressing surface area increased upto 79.9% by SAAT treatment for 10 sec. Other tissues that are considered difficult to transform can be subjected to SAAT that permit Agrobacterium to infect deep within the plant tissue. While SAAT treatment was not found effective at post co-cultivation period with decreased shoot proliferation from cotyledonary node of some soybean genotypes (Meurer et al., 1998). They also reported that inoculum OD600 1.0 gave better transient expression but no interaction was found between SAAT, *Agrobacterium* strain and soybean genotype. Micro-wound in plant tissues due to SAAT treatment release compounds that facilitate growth and accumulation of bacteria under aerobic conditions so facilitate transformation efficiency (Finer & Finer, 2000). However, longer sonication time may damage plant tissue (Santarem et al. 1998). Way of placement of explant (adaxial side incontact with medium) on medium (Ko et al., 2003); exposure of soybean explants to AgNO3 throughout shoot induction and shoot elongation (Olhoft et al., 2004); explant preparation in the presence of *Agrobacterium* culture and varying level of kanamycin during selection and regeneration (Zia et al., 2010b) are important for better recovery of

Instead of kanamycin resistant plant, glufosinate resistant (bar gene) plants were produced by Zhang et al. (1999) and Clemente et al. (2000) using cotyledonary node explants of 5 days old seedlings. Glyfosinate selection regime is important to get rid of non-transformed plants and to minimize chimerism. Yan et al. (2000) analyzed that immature zygotic size 8-10mm and co-cultivation for short period increase transient expression while selection by direct replacement at low concentration of hygromycin also increase somatic embryo development and plant regeneration. Cystine present in co-cultivation medium increase transformation efficiency due to presence of thiol group and polyphenol oxidase and peroxidase inhibition (Olhoft & Somers, 2001; Olhoft et al., 2001). Copper and iron chelators were also found effective for better expression. Olhoft and his colleagues successfully transformed soybean by cot node method (Olhoft et al., 2003). High frequency upto 16.4% was observed due to presence of cystine, Dithiothreitol (DTT) and thiol compound in infection and co-cultivation medium. Beside this, addition of Silwet-77 as surfactant; co-cultivation at 22C also played significant role in transformation (Liu et al., 2007). Donaldson & Simmonds (2000) demonstrated that competent cells, in the case of cotyledonary node transformation, are few so has low transformation competency therefore using cotyledonary nodes as explants present low transformation efficiency. Tight selection procedure (selection of explants on selective agent before infection) increases transformation efficiency and occurrence of less

Xing and his colleagues produced marker free plants by introducing two T-DNA binary systems (Xing et al., 2000). Integration of two T-DNA followed by their independent

transformants.

escape (Chen, 2004).

Engineering technologies and molecular mindsets expiated asset of *Agrobacterium* to transfer the genes of interest into plant cell. This revolution lighted the pathway to break inert kingdom genetic exchange restrictions. They terminated the property of *Agrobacterium* to cause tumor but did not change the belongings that are involved in T-DNA transfer mechanism. Finally, plant biotechnological era came to revolution to produce transgenic plant species with desired characters. However, all the barriers could not be departed productively. Factors responsible for production of transformants have been studies worldwide and are found more or less same for all genotypes even plant or Agrobacterium. These factors, at *Agrobacterium* flank, include genotype, plasmid constrains, T-DNA length and signaling mechanism. While at plant cell side, the factors include type, age, genetic makeup and welcome address to T-DNA. The welcome discourse also depends upon physical and chemical conditions that finally lead to produce whole plant from a single transformed cell.

Although initially dicots were considered host for *Agrobacterium* but advancement in procedures commanded *Agrobacterium* to display same role in monocots as in dicots. The process of plant transformation is a routine matter in most of the labs but some plant species are still considered recalcitrant to transformation.
