<div>Background: Common bean (Phaseolus vulgaris L.) is a globally important grain and vegetable legume crop, providing a substantial portion of the diet protein and minerals for many people in the developing world. However, the genetic studies and improvement on this crop has long been impeded by its recalcitrance to Agrobacterium-mediated whole plant genetic transformation. Established Agrobacterium rhizogenes-based hairy root transformation in common bean heavily relies on the strain K599. 
 
Methods: In order to develop an efficient alternative protocol for hair transformation in common bean, the efficiency of Agrobacterium rhizogenes strain R1000 in inducing hairy roots from 6-day-old seedlings with root below cotyledons excised by the soaking and smearing method were tested. The binary plasmid pBI121 with the reporter gene GUS (pBI121-GUS) or eGFP (pBI121-eGFP) driven by the constitutive promoter was used for transformation and rapid identification of the transgenic hairy roots.&nbsp; &nbsp; 
 
Result: We established a strain R1000-based system for the induction of hairy roots in common bean. The plant receptor genotypes and infection methods were optimized, which led to a high transformation rate of hairy roots up to 60%. This method therefore provides a useful alternative means for functional genomic studies in common bean.</div>

​​An Agrobacterium rhizogenes Strain R1000-mediated Efficient Hairy Root Transformation Protocol for Common Bean

<div>Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Oxalis corniculata evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Five fold transformation efficiency is achieved in transient gus gene expression after optimizing various parameters. The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (400 &mu;M) and cocultivation temperature of 22&deg;C. 
 
Methods: Oxalis corniculata (L.) were used for&nbsp; transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56,0.80 and 1.34 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of BAP 2 mg L-1 and NAA 0.5 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400,600 and 800 &mu;M. Histochemical analysis of gene expression was carried out. 
 
Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 64% when the infection process was carried out with acetosyringone in co-cultivation medium (400 &mu;M). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.</div>

Optimization of Agrobacterium tumefaciens-mediated Transformation in Oxalis corniculata (L.)

<div>Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Euphorbia tirucalli evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Optimized parameters resulted in high transformation efficiency 3 fold increase at transient GUS expression .The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (100 &micro;M) and cocultivation temperature of 25&deg;C. 
 
Methods: Nodal explants were used for&nbsp; transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56, 0.8 and 1.2 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of NAA 2 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400, 600 and 800 &micro;M. Histochemical analysis of gus gene expression was carried out. 
 
Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 72% when the infection process was carried out with acetosyringone in co-cultivation medium (100 &micro;M). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.</div>

An Attempt to Establish an Agrobacterium-mediated Transient Expression in Euphorbia tirucalli (L.) an Important Medicinal Plant

<div>Background: Green gram is grown in many parts of India as a source of dietary protein (21-25%). It is an important nitrogen fixing crop which fixes atmospheric nitrogen (119-140 kg/ha) to soil and enhance the soil productivity. In the present investigation, efficient Agrobacterium-mediated genetic transformation of Vigna radiata L. (Wilczek) has been achieved with VrTIP1 gene for abiotic stress resistance i.e. moisture and salinity stress. 
 
Methods: Four days old shoot tip and cotyledonary node were used for in vitro regeneration with MS medium supplemented with BAP 2.0 mg/l, kinetin 0.5 mg/l and 50 mg/l kanamycin for co-cultivation with Agrobacterium tumefaciens strains, LBA 4404. The modified binary vector pCXSN, EHA105 containing hygromycin phosphotransferase II (hpt II) marker genes and a synthetic TIP1 gene under a constitutive CaMV35S promoter were used for transformation of Vigna radiata L. cotyledonary node explants. Putative transformants selected from hygromycin resistant shoots were subsequently rooted on MS medium supplemented with 1.0 mg/l NAA and later transferred to sterile vermiculite followed by transfer to the transgenic green house. 
 
Result: The T1 plants were produced from PCR positive T0 plants and analysed for presence and integration of transgenes in putative T1 plants were confirmed by polymerase chain reaction (PCR) amplification of 752 bp of hpt II fragment. This protocol can be effectively used for transferring new traits in greengram and other legumes for their quantitative and qualitative improvements.</div>

Agrobacterium tumefaciens-Mediated Genetic Transformation of Green Gram [Vigna radiata (L.) Wilczek] - A Recalcitrant Grain Legume

An efficient Agrobacterium mediated transformation system, from which transgenic tropical maize plants was directly generated without previous crosses with laboratory or temperate lines was established in present study. Experimental evaluations are focused on two main issues: i) Establishment of appropriate tissue culture conditions, which induced somatic embryogenesis from the immature embryo cells, and ii) The delivery of T-DNA toward these cells. &nbsp;A high rate of embryogenic calli, generated from immature embryo was obtained when 2-4 D (4mgL-1) was added to the MS based induction medium. Regeneration rate was observed upto 19 plants per gram in the calli by using BAP and KIN at 0.5mgL-1. &nbsp;Regarding the Agrobacterium strains evaluated for their transformation capability on the tropical maize line MU-2092 used, best results are obtained from the strain when applied at OD 550nm= 0.5-1.0. Physical micro-wounds before the Agro-infection proved to be an excellent way to promote both the T-DNA transfer toward the immature embryo and the acceleration in rates of transient GUS expression when compared with particle bombardment. The highest frequencies of transient GUS expression corresponding to the embryos as well as the regeneration of whole transgenic plants emerged from them, was obtained using immature embryos wounded by bombarding at 80 lb/in2. Analysis of the progenies confirmed the sexual transmission of the introduced genes and their stable expression.

An efficient regeneration and genetic transformation of maize through Agrobacterium and particle bombardment in immature embryos 
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<div>Background: Agrobacterium mediated genetic transformation experiments were carried out in Dolichos bean Cv. (Konkan Bhushan) showing better regenerability.&nbsp; 
 
Methods: Three cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa were used for the transformation each of which were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. A vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 was used. Mature embryo axis with single cotyledon was used as explant. Kanamycin as well as PCR screening was carried out to assess the transformation frequency. Progeny analysis using PCR was also carried out to assess the transgene segregation and stable transformation. 
 
Result: Kanamycin concentration of 500 mg/l was found as optimum for selection of a transgenic turning leaf blades albino. Among five methods of colonization used, the method employing mild injury to explant with dipping in Agrobacterium culture for 20 minutes followed by co-cultivation for 48 hours, cefotaxime washing and sowing in soil resulted in maximum survival (74.80%) associated with maximum transformation frequency through PCR analysis (2.13%). Among three cry genes, the gene cry2Aa was found the most effective in transforming Dolichos bean. The progeny analysis of transformants has shown the 3:1 mendelian segregation ratio confirming stable transformation of transgene.</div>

<div>Genetic Transformation for Pod Borer Resistance in Dolichos Bean [Lablab purpureus (L.) Sweet]</div>

Background: Common bean (Phaseolus vulgaris L.) is a major food legume with high nutritional value and economic importance globally. A remarkable attribute of this plant is its propensity to generate adventitious roots from petioles of detached leaves when moisture is appropriate. This distinctive feature presents promising prospects for harnessing petioles in the facilitation of transgenic hairy root production. 
 
Methods: We achieved the successful induction of transgenic hairy roots from common bean petioles through the utilization of Agrobacterium rhizogenes strain K599. Our experimentation encompassed a diverse array of common bean varieties and leaves of varying ages. Subsequently, we refined and optimized the procedure for hairy root induction.&nbsp; 
 
Result: Our investigations have revealed that the most conducive conditions for hairy root transformation with K599 are attained using 5-day-old leaves from the cultivar &lsquo;Honghuaqingjia&rsquo;, exhibiting a remarkable induction efficiency of 59%. The usefulness of this system was demonstrated through a subcellular localization analysis of the transcription&nbsp;factor PvTCP2 protein in combination with GFP (Green Fluorescent Protein).

An Efficient Hairy Root Transformation Method for Common Bean based on Petiole Explants

<div style="text-align: justify;">Decades of documented successful pieces of evidence in agritech have shown us a clear picture of the importance of biotechnology in crop improvement. The production in agriculture should have steady growth and to achieve these objective conventional methods should go on parallel with biotechnological approaches. Genetic engineering which has revolutionized the path of crop improvement involves the identification and transfer of novel genes into the existing elite cultivars. Different methods of transferring the gene into plant cells have been developed and continuous efforts have been made to increase its efficiency. Both direct and indirect method of gene transfer has its own merits and demerits. Efforts have been made continuously to eliminate drawbacks and to develop an easy, elite and eco-friendly method to transfer genes. The transformation method which is a base of genetic engineering is vital and Agrobacterium-mediated gene transfer and gene gun have shown to be doing well in recent years. As a whole the methodology involved, merits and demerits of different methods have been briefly discussed.</div>

<div style="text-align: justify;">Genetic Transformation Methods for Crop Improvement- A Brief Review</div>

<div>Background: Salinity is a major problem worldwide and is increasing day by day. Salt stress causes severe yield losses in crop plants and the damages in chickpea are up to 100%. To overcome these losses, the present study was undertaken to develop transgenic chickpea plants (var. HC-1) carrying OsRuvB gene for salt stress tolerance. 
 
Methods: Transgenic chickpea plants harboring OsRuvB gene were developed using Agrobacterium-mediated transformation. T0 putative transgenic chickpea plants were screened for the presence of OsRuvB gene through PCR using gene specific primers. The stable integration and copy number of transgene in transgenic chickpea plants were confirmed through Southern hybridization and qRT-PCR. T1 generation transgenic chickpea plants were screened for the presence of OsRuvB gene using direct PCR (Phire Direct PCR kit). 
 
Result: PCR-based screening of putative transformants using gene-specific primers showed a transformation frequency of 17%. Southern blot and real-time PCR analysis revealed stable and single-copy insertion. In T1 generation a total of 74 plants (out of 170) showed the presence of OsRuvB gene. The engineered lines developed in the present investigation can be further undertaken to develop transgenic chickpea plants for salt stress tolerance.</div>

Engineering Chickpea Variety HC-1 with OsRuvB Gene for Salt Stress Tolerance

<div style="text-align: justify;">In planta transformation has been established as an innovative and simple technique involving direct transformation of plant parts without involving the tedious tissue culture step. Methodologies of in planta transformation involve different plant parts and strategies. Agrobacterium strain carrying the gene of interest is targeted to the specific plant part either directly or in the induction medium. Vacuum infiltration is sometimes used to facilitate foreign gene integration. Initial studies of this novel technique involved treatment of whole plants with the inoculum, and later shifted to treatments of shoot tips and floral parts and finally the female reproductive parts have been targeted. Zygotes, embryos and seeds have recently been used extensively yielding successful transformation. The method is simple, convenient and overcomes the problem of tissue culture induced genetic variability in the transformants. The review traces the origin and development in the in planta methodologies used over the past and the various parameters considered by the workers for increased effectiveness viz. developmental stages, Agrobacterium stain, surfactant, induction medium etc., in the various crops compiled. Based on the review it may be inferred that there is immense potential in planta transformation, and the ease of regeneration and selection of transformants in the methods described, can be utilized for crop improvement.</div>