Application of sonication in combination with vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars.

Soybean is a recalcitrant crop to Agrobacterium-mediated genetic transformation. Development of highly efficient, reproducible, and genotype-independent transformation protocol is highly desirable for soybean genetic improvement. Hence, an improved Agrobacterium-mediated genetic transformation protocol has been developed for cultivar PK 416 by evaluating various parameters including Agrobacterium tumefaciens strains (LBA4404, EHA101, and EHA105 harboring pCAMBIA1304 plasmid), sonication duration, vacuum infiltration pressure, and vacuum duration using cotyledonary node explants of soybean prepared from 7-day-old seedlings. The transformed plants were successfully developed through direct organogenesis system. Transgene expression was assessed by GUS histochemical and gfp visual assays, and integration was analyzed by PCR and Southern blot hybridization. Among the different combinations and durations evaluated, a maximum transformation efficiency of 18.6 % was achieved when the cotyledonary node explants of cv. PK 416 were sonicated for 20 s and vacuum infiltered for 2 min at 250 mmHg in A. tumefaciens EHA105 suspension. The amenability of the standardized protocol was tested on four more soybean cultivars JS 90-41, Hara Soy, Co 1, and Co 2 in which all the cultivars responded favorably with transformation efficiency ranging from 13.3 to 16.6 %. The transformation protocol developed in the present study would be useful to transform diverse soybean cultivars with desirable traits.

Assessment of somatic embryogenesis potency in Indian soybean [Glycine max (L.) Merr.] cultivars.

Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication reports the development of somatic embryogenesis in a liquid culture medium from immature cotyledons of G. max. Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic embryos and percentage of explants that responded were higher with 45.24 microM 2,4-D. The proliferation of somatic embryos for three successive cycles was achieved in 22.62 microM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 microM) indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L-asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with 74.42% of response.

Overexpression of tobacco osmotin (Tbosm) in soybean conferred resistance to salinity stress and fungal infections.

Salinity and fungal diseases are the two significant constraints limiting soybean productivity. In order to address these problems, we have transformed soybean cv. Pusa 16 via somatic embryogenesis with salinity induced and apoplastically secreted pathogenesis-related tobacco osmotin (Tbosm) gene using Agrobacterium-mediated genetic transformation. Integration of Tbosm in randomly selected five GUS assay-positive independently transformed soybean plants was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Reverse transcriptase-PCR (RT-PCR) and Western blotting confirmed that the Tbosm was expressed in three of the five transformed soybean plants. Further the Western blotting revealed that the truncated osmotin protein accumulated more in apoplastic fluid. The transformed (T(1)) soybean plants survived up to 200 mM NaCl, whereas non-transformed (NT) plants could withstand till 100 mM and perished at 150 mM NaCl. The biochemical analysis revealed the T(1) soybean plants accumulated higher amount of proline, chlorophyll, APX, CAT, SOD, DHAR, MDHAR, and RWC than NT plants. Leaf gas exchange measurements revealed that T(1) soybean plants maintained higher net photosynthetic rate, CO(2) assimilation, and stomatal conductance than NT plants. The three T(1) soybean plants expressing the osmotin gene also showed resistance against three important fungal pathogens of soybean--Microsphaera diffusa, Septoria glycines and Phakopsora pachyrhizi. The T(1) soybean plants produced 32-35 soybean pods/plant containing 10.3-12.0 g of seeds at 200 mM NaCl, whereas NT plant produced 28.6 soybean pods containing 9.6 g of seeds at 100 mM NaCl. The present investigation clearly shows that expression of Tbosm enhances salinity tolerance and fungal disease resistance in transformed soybean plants.

Antioxidant effect of dietary supplement Withania somnifera L. reduce blood glucose levels in alloxan-induced diabetic rats.

The phenolic compounds and flavonoids were determined from the extracts of Withania somnifera root (WSREt) and leaf (WSLEt). The WSREt has 28.26 mg/g total phenolic compounds and 17.32 mg/g flavonoids, whereas WSLEt has 5.4 mg/g total phenolic compounds and 5.1 mg/g flavonoids. The WSREt, WSLEt and glibenclamide were orally administered daily to diabetic rats for 8 weeks. After the treatment, the levels of urine sugar, blood glucose, liver glycogen, and antioxidants like vitamin C and E in plasma and superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and reduced glutathione (GSH) in liver, kidney and heart were determined. Diabetic rats showed a significant (p < 0.05) elevation in glucose and TBARS and a significant (p < 0.05) reduction in glycogen, vitamin C and E, SOD, CAT, GPx, GST, and GSH levels when compared to normal control rats. Administration of WSREt, WSLEt and glibenclamide to diabetic rats restored the levels to normal. In the light of aforesaid facts, it is suggested that the presence of phenolic compounds including flavonoids in W. somnifera root and leaf extracts and their antioxidant activity may play a vital role in reduction of blood glucose level in alloxan-induced diabetic rats.

Hypoglycaemic and hypolipidaemic effects of Withania somnifera root and leaf extracts on alloxan-induced diabetic rats.

Withania somnifera is an important medicinal plant, which is used in traditional medicine to cure many diseases. Flavonoids were determined in the extracts of W. somnifera root (WSREt) and leaf (WSLEt). The amounts of total flavonoids found in WSREt and WSLEt were 530 and 520 mg/100 g dry weight (DW), respectively. Hypoglycaemic and hypolipidaemic effects of WSREt and WSLEt were also investigated in alloxan-induced diabetic rats. WSREt and WSLEt and the standard drug glibenclamide were orally administered daily to diabetic rats for eight weeks. After the treatment period, urine sugar, blood glucose, haemoglobin (Hb), glycosylated haemoglobin (HbA1C), liver glycogen, serum and tissues lipids, serum and tissues proteins, liver glucose-6-phosphatase (G6P) and serum enzymes like aspartate transaminase (AST), alanine transaminase (ALT), acid phosphatase (ACP) and alkaline phosphatase (ALP) levels were determined. The levels of urine sugar, blood glucose, HbA1C, G6P, AST, ALT, ACP, ALP, serum lipids except high density lipoprotein-bound cholesterol (HDL-c) and tissues like liver, kidney and heart lipids were significantly (p < 0.05) increased, however Hb, total protein, albumin, albumin:globulin (A:G) ratio, tissues protein and glycogen were significantly (p < 0.05) decreased in alloxan-induced diabetic rats. Treatment of the diabetic rats with WSREt, WSLEt and glibenclamide restored the changes of the above parameters to their normal level after eight weeks of treatment, indicating that WSREt and WSLEt possess hypoglycaemic and hypolipidaemic activities in alloxan-induced diabetes mellitus (DM) rats.