SONICATION AND VACUUM INFILTRATION ENHANCED AGROBACTERIUM RHIZOGENES MEDIATED TRANSFORMATION IN SOYBEAN

AbstractObjective: The present study involved the formulation of protocol of Agrobacterium rhizogenes-mediated transformation for the detailed study of isoflavones metabolism in soybean.Methods: Cotyledons were separated from 4-day-old soybean seedlings and infected with three different A. rhizogenes strains under various time durations of sonication, vacuum infiltration and co-cultivated on MS medium supplemented with various concentrations of acetosyringone. The induced hairy roots were established as a culture with the selection agent hygromycin B. Transgenes integrated in hairy roots were analysed at molecular level by PCR assay.Results: A. rhizogenes strain R1000 harbouring pCAMBIA1301 resulted in better transformation efficiency when compared with other strains. The optimum duration of sonication (2 min) and vacuum infiltration (2 min) enhanced the transformation efficiency up to 76.47 %.  PCR analyses revealed the integration of transgene in hairy roots lines.Conclusion:  From the present study, we could conclude that, sonication and vacuum infiltration techniques could be employed to produce genotype independent transgenic soybean hairy root lines and which could be used to study for the improved production of potent anti-cancer compounds, isoflavones in soybean.Keywords: Agrobacterium, isoflavones, soybean, sonication, vacuum infiltratio

Assessment of factors influencing the tissue culture-independent Agrobacterium-mediated in planta genetic transformation of okra [Abelmoschus esculentus (L.) Moench]

Regeneration of transformed plants from the Agrobacterium-infected tissue is a time-consuming process and requires hard work. Okra [Abelmoschus esculentus (L.) Moench] is highly recalcitrant to Agrobacterium-mediated genetic transformation and regeneration. In this study, we established a tissue culture-independent genetic transformation system for okra using seed as an explant. Agrobacterium tumefaciens EHA 105 harbouring the binary vector pCAMBIA 1301-bar was used to infect the okra seeds. Various parameters influencing the okra genetic transformation including, co-cultivation duration, acetosyringone, sonication, and vacuum infiltration have been evaluated. Maximum transformation efficiency of 18.3 % was recorded when the pre-cultured okra seeds were sonicated for 30 min and vacuum infiltrated for 3 min in Agrobacterium suspension containing 100 A mu M acetosyringone and co-cultivated for 3 days on a medium containing 100 A mu M acetosyringone. The GUS histochemical analysis confirmed the gus A gene integration and expression, whereas polymerase chain reaction (PCR) and Southern blot hybridization confirmed the bar gene integration and copy number in the transformed okra genome. The transgene was successfully segregated into the progeny plants with a Mendelian inheritance ratio of 3:1. The in planta transformation protocol developed in the present investigation is applicable to transform the okra plants with disease-resistant traits, and the transformed plants can be generated within 60 days

Productivity of withanolides in shake-flask culture and bioreactor by elicitor and precursor treatments in cell suspension of <i>W. somnifera</i>.

<p>For Shake-flask culture.</p><p>Initial inoculum – ∼500 mg cell mass for 30 ml MS medium.</p><p>For Bioreactor culture.</p><p>Initial inoculum – ∼83 g FW cell mass for 5 l MS medium.</p><p>Data were recorded on 28^th day of culture for both the experiments.</p><p>Values represent the mean ± standard error of three experiments. Mean values followed by the same letters within a column are not significantly different according to Duncan's multiple range test at 5% level.</p

Agrobacterium-mediated in planta genetic transformation of sugarcane setts

An efficient, reproducible, and genotype-independent in planta transformation has been developed for sugarcane using setts as explant. Traditional Agrobacterium-mediated genetic transformation and in vitro regeneration of sugarcane is a complex and time-consuming process. Development of an efficient Agrobacterium-mediated transformation protocol, which can produce a large number of transgenic plants in short duration is advantageous. Hence, in the present investigation, we developed a tissue culture-independent in planta genetic transformation system for sugarcane using setts collected from 6-month-old sugarcane plants. The sugarcane setts (nodal cuttings) were infected with three Agrobacterium tumefaciens strains harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA(A (R)). Several parameters influencing the in planta transformation such as A. tumefaciens strains, acetosyringone, sonication and exposure to vacuum pressure, have been evaluated. The putatively transformed sugarcane plants were screened by GUS histochemical assay. Sugarcane setts were pricked and sonicated for 6 min and vacuum infiltered for 2 min at 500 mmHg in A. tumefaciens C58C1 suspension containing 100 A mu M acetosyringone, 0.1 % Silwett L-77 showed the highest transformation efficiency of 29.6 % (with var. Co 62175). The three-stage selection process completely eliminated the chimeric transgenic sugarcane plants. Among the five sugarcane varieties evaluated using the standardized protocol, var. Co 6907 showed the maximum transformation efficiency (32.6 %). The in planta transformation protocol described here is applicable to transfer the economically important genes into different varieties of sugarcane in relatively short time

Enhanced Biosynthesis of Withanolides by Elicitation and Precursor Feeding in Cell Suspension Culture of <i>Withania somnifera</i> (L.) Dunal in Shake-Flask Culture and Bioreactor

<div><p>The present study investigated the biosynthesis of major and minor withanolides of <i>Withania somnifera</i> in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28^th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.</p></div

Growth kinetics and withanolides production in cell suspension culture of <i>W. somnifera</i>.

<p>a. Dynamic profiles of biomass accumulation, b. Dynamic profiles of withanolides production, c. Synchronized cell suspension culture in 150-flask culture, d. Lab scale cell suspension culture in 7-l bioreactor. The cell suspension cultures were established by inoculating ∼500 mg fresh mass of friable callus in 150 ml Erlenmeyer flask containing 30 ml of MS liquid medium supplemented with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose and kept on gyratory shaker at 120 rpm under total darkness. Bioreactor culture was established with 83 g FW of friable callus in 5-l MS liquid medium with same hormonal combinations. Values represent mean ±standard error of three replicates; each experiment was repeated thrice. All bars = 1 cm.</p

Effect of carbon and nitrogen sources on <i style="mso-bidi-font-style:normal">in vitro</i> flower and fruit formation and withanolides production in <i style="mso-bidi-font-style:normal">Withania somnifera</i> (L.) Dunal

177-183<span style="mso-ansi-language: FR" lang="FR">We studied the influence of sucrose and nitrogen concentration on in vitro flowering and fruit setting in elongated shoots of Withania somnifera. BA (1.5 mg/l) and IAA (0.3 mg/l) on MS medium supplemented with 4% sucrose showed 67% of in vitro flower induction frequency, 9 flowers/shoot, 4 fruits/shoot and 11 seeds/fruit in elongated-shoots. Different concentrations of nitrogen sources (L-glutamine, adenine sulphate, ammonium nitrate, potassium nitrate and sodium nitrate 5-25 mg/l) were tested in combination with 4% sucrose and BA at 1.5 mg/l and IAA at 0.3 mg/l. Highest number of flowers (20 flowers/shoot; 2.2-fold) and fruits (16 fruits/shoot; 3.39-fold), fruit setting (12 seeds/fruit; 1.08-fold) at a higher frequency (88 %) were achieved on MS medium augmented with 15 mg/l adenine sulphate with same PGRs and sucrose concentration. The maximum production of withanolide A (0.68 mg/g DW) and withanolide B (0.77 mg/g DW) was recorded in in vitro fruits. Highest accumulation of withaferin A (2 mg/g DW) was quantified from <i style="mso-bidi-font-style: normal">in vitro flowers, whereas, it was low in <i style="mso-bidi-font-style: normal">in vitro fruits (0.49 mg/g DW withaferin A). However, withanone (0.23 mg/g DW) was found accumulated uniformly in both <i style="mso-bidi-font-style: normal">in vitro flowers and fruits compared to control. </span

Highly efficient Agrobacterium-mediated in planta genetic transformation of snake gourd (Tricosanthes cucumerina L.)

Regeneration of plants from Agrobacterium tumefaciens infected cell or tissue is a time-consuming process and somaclonal variations are the major bottleneck. Hence, in the present study, a tissue culture-independent genetic transformation system for snake gourd was developed using seed as an explant. Snake gourd seeds were infected with three A. tumefaciens strains harbouring pGA 492 plasmid, and the transformants were selected against BASTA(A (R)). Several factors influencing the in planta genetic transformation such as Agrobacterium strains, acetosyringone, sonication, and vacuum infiltration, have been evaluated. The maximum transformation efficiency (19.6 %) was recorded when the pre-cultured snake gourd seeds were sonicated for 30-min and vacuum infiltrated for 3-min at 750 mm of Hg in A. tumefaciens EHA 105 suspension containing 100 A mu M of acetosyringone. The bar gene integration into the snake gourd genome was confirmed by polymerase chain reaction and Southern blot hybridization. The transgene was successfully inherited into the T-1 progeny plants. This transformation method can produce transgenic snake gourd plants in a relatively short time (35-days)

The effect of different concentrations of mevalonic acid on biomass accumulation (a) and withanolides production (b) in cell suspension culture of <i>W. somnifera</i> in shake-flask culture at 48 h exposure time.

<p>Five hundred milligram of fresh mass of friable callus was cultured in 30/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose and kept on gyratory shaker at 120 rpm under total darkness. The cultures were harvested on 28^th day. Data represents mean±standard error of three replicates; each experiment was repeated thrice.</p

Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal.

In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants