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

Role of polyamines on in vitro regeneration and podophyllotoxin production in Podophyllum hexandrum Royle

Podophyllum hexandrum is a popular medicinal plant endemic to Himalayas. In the present investigation, we report an efficient mass propagation protocol and Podophyllotoxin (PTOX) production in in vitro-derived plants of P. hexandrum, cultured on MS medium containing various plant growth regulators and polyamines. A combination of BA (1.5 mg/L), IAA (0.2 mg/L) and spermidine (20 mg/L) produced the highest number of multiple shoots per explant (23 shoots) after 6 weeks of culture; the regenerated shoots were elongated on the same medium. The elongated-shoots were rooted on root induction medium (half strength MS medium) supplemented with IBA (1.5 mg/L) and putrescine (15 mg/L). The rooted plantlets were successfully hardened and acclimatized with a survival rate of 68% in the greenhouse. The highest content of PTOX (4.23 mg/g DW) was recorded in in vitro derived roots followed by leaves when compared to field-grown parent plants. The present system offers the possibility to use in vitro culture techniques for mass propagation and PTOX production for commercial utilization

Role of polyamines on in vitro regeneration and podophyllotoxin production in Podophyllum hexandrum Royle

777-787Podophyllum hexandrum is a popular medicinal plant endemic to Himalayas. In the present investigation, we report an efficient mass propagation protocol and Podophyllotoxin (PTOX) production in in vitro-derived plants of P. hexandrum, cultured on MS medium containing various plant growth regulators and polyamines. A combination of BA (1.5 mg/L), IAA (0.2 mg/L) and spermidine (20 mg/L) produced the highest number of multiple shoots per explant (23 shoots) after 6 weeks of culture; the regenerated shoots were elongated on the same medium. The elongated-shoots were rooted on root induction medium (half strength MS medium) supplemented with IBA (1.5 mg/L) and putrescine (15 mg/L). The rooted plantlets were successfully hardened and acclimatized with a survival rate of 68% in the greenhouse. The highest content of PTOX (4.23 mg/g DW) was recorded in in vitro derived roots followed by leaves when compared to field-grown parent plants. The present system offers the possibility to use in vitro culture techniques for mass propagation and PTOX production for commercial utilization

meta-Topolin and β-cyclodextrin enhance multiple shoot and root production in black gram Vigna mungo (L.) Hepper

314-322The recalcitrant nature of black gram is the major constraint of in vitro regeneration and agrobacterium- mediated genetic transformation, to overcome this, a productive shoot regeneration protocol has been achieved in black gram cultivar T9 using 7-day old cotyledonary node explants excised from in vitro- raised black gram seedlings using meta-topolin. An aromatic cytokinin, meta-topolin along with BA (1.5+0.5 mg/L) in 0.50 strength MS medium with 1.5% (w/v) sucrose exhibited a maximum number of multiple shoots (32.0±0.37 shoots/explant) at the end of 6 weeks of culture. The shoots were elongated in (6.40±0.50 cm/shoot) in MS medium supplemented with GA3 (2.0 mg/L). A maximum number of roots (9.60±0.50/shoot) and root length (11.20±0.73 cm/shoot) were obtained in combination with β-cyclodextrin (a cyclic oligosaccharide; 1.5 mg/L) and IBA (1.5 mg/L). The rooted plantlets were hardened and acclimatized with least mortality rate of 2% in pot mixture consisting red soil:sand:farm yard manure (FYM) (2:1:1) and grown in green house with 85% relative humidity. Ploidy levels were analyzed using flow cytometry which confessed the chromosomal stability in invitro raised plants similar to parent plants. This protocol may be useful for producing transgenic black gram with desirable agro-traits in Indian cultivars

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

Determination of Mineral Content in Methanolic Safflower (Carthamus tinctorius L.) Seed Extract and Its Effect on Osteoblast Markers

Safflower (Carthamus tinctorius L.) seeds are used as a folk medicine to enhance bone formation or to prevent osteoporosis in Korea. Therefore, the methanolic extract of safflower seeds (MESS) containing high mineral content, such as calcium (Ca), potassium (K) and phosphorous (P), was evaluated for the role on osteoblast (Ob) markers of Sprague-Dawley rats. In serum of 3 to 11 weeks (wks) old rats, both osteocalcin (OC) content and bone-specific alkaline phosphatase (B-ALP) activity increased to their maximum levels in 4–7 wks. Hence, 3 wks old rats were selected for 8 wks oral treatment of MESS, resulted in the significant increase of Ob markers in serum such as OC content (4–8 wks), B-ALP activity (1–2 wks) and insulin-like growth factor I (IGF-I) level (1 wk), and the growth parameter such as the length of femur (2–8 wks) and tibia (4 wks). On the basis of Pearson’s correlation coefficient, there were a moderate correlation between OC and B-ALP at 8 wks, a low correlation between OC and IGF-I at 1, 4 and 8 wks, a moderate correlation between OC and femur length at 1, 2 and 8 wks, and a moderate correlations between OC and tibia length at 1 and 8 wks of MESS-treated groups. The result reveals that the changes of OC correlated at low to moderate level with the changes of B-ALP activity, IGF-I content and femur and tibia length in the MESS-treatment period. On the other hand, there were a strong correlation between IGF-I and femur length at 2 wks and moderate correlation between IGF-I and tibia length at 1, 2 and 8 wks of MESS-treated groups. Therefore, the effect of MESS on bone formation likely appears to be mediated by IGF-I at the early stage of treatment

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