03263nas a2200277   4500008004100000022001400041245017200055210006900227260000800296300001200304490000800316520228800324653002402612653001402636653002602650653002602676653000902702653001502711653002202726100002302748700002002771700001902791700002002810700002802830856012702858       2018                        eng d  a0926-669000aDevelopment of enhanced hypericin yielding transgenic plants and somaclones: high throughput direct organogenesis from leaf and callus explants of Hypericum perforatum0 aDevelopment of enhanced hypericin yielding transgenic plants and  cJAN  a544-5540 v1113 a<p>Hypericum perforatum is well known for its anti-depressent properties due to the presence of hypericins and hyperforins. The direct regeneration protocol from leaf explants and Agrobacterium tumefaciens mediated genetic transformation is a prerequisite for gene transfer studies in this valuable plant system as H. perforatum has shown its recalcitrance to these studies previously. The aim of generating transgenic shoots or somaclones in the present study is to select high hypericine yielding H. perforatum plants. High throughput direct bud organogenesis from leaf explants has been achieved for the first time in this plant system on media combination of MS + 5.0 mg/l BAP + 1.0 mg/l. This medium interestingly also worked as the indirect regeneration media for generation of somaclones from callus cultures. Thus far optimized single media can be used for direct and indirect regeneration in H. perforatum. The direct regeneration frequency of plantlets from leaf explants was found to be 80% while 100% was the regeneration frequency noted for indirect plantlet regeneration from callus cultures. The optimized direct regeneration protocol from leaf explants was utilized for A. tumefaciens (harbouring pCAMBIA 1301) mediated genetic transformation studies with the aim of establishing transgenic lines of H. perforatum. The resultant study successfully gave rise to kanamycin resistant GUS positive shoots with transformation efficiency of 3136 +/- 3.6. A total of four transgenic H. perforatum clones namely T2, T3, T5 and T8 with different growth parameters were established, where highest of fivefold increase in hypericin content was recorded by T5 (276.8 +/- 9.2 mu g/g dry wt.) in comparison to control non transformed plants (35.6 +/- 2.7 mu g/g dry wt.). Interestingly the presence of serotonin and melatonin was also detected in transgenic plants on TLC basis. The transgenic nature of the plants was confirmed by PCR with amplification of uidA gene. Apart from it, from indirect regeneration from callus, total of ten somaclones were generated. The ISSR profiling of somaclones scored a total of 156 bands, among which 118 were polymorphic in nature. The glasshouse established soma clones showed up to twofold increase in hypericin content on HPLC analysis.</p>10aDirect regeneration10aHypericin10aHypericum perforation10aIndirect regeneration10aISSR10aSomaclones10aTransgenic plants1 aKhan, Shamshad, A.1 aVerma, Priyanka1 aArbat, Akshata1 aGaikwad, Sushma1 aParasharami, Varsha, A.  uhttp://library.ncl.res.in/content/development-enhanced-hypericin-yielding-transgenic-plants-and-somaclones-high-throughput02919nas a2200253   4500008004100000022001400041245010000055210006900155260000800224300001200232520206200244653003102306653002002337653001602357653002802373653001102401653001402412653002502426653001602451100002602467700002002493700002802513856012402541       2022                        eng d  a0167-685700aHomo and heterologous expression of the HpPKS2 gene in Hypericum perforatum and Bacopa monnieri0 aHomo and heterologous expression of the HpPKS2 gene in Hypericum  cJAN  a215-2153 a<p>Hypericum perforatum has been known to produce hypericin and hyperforin that are used in treating mild to moderate depression. The HpPKS2 (H. perforatum polyketide synthase 2) gene is assumed to play a role in hypericin biosynthesis. The HpPKS2 gene was overexpressed in homologous H. perforatum in vitro grown plants through Agrobacterium tumefaciens-mediated genetic transformations. It leads to the establishment of seven glass house acclimatized transgenic lines. Among them, the HP12 transgenic plant showed 9.8 fold enhancement in hypericin content (379.4 +/- 10.3 mu g/g dry wt) followed by 3-fold in HP41 (117.1 +/- 4.5 mu g/g dry wt) as compared to control plants. This was further supported by the real-time PCR studies where it registered up to 5 fold enhancement of HpPKS2 gene expression. On the other hand, the heterologous expression of the HpPKS2 gene in Bacopa monnieri resulted in the establishment of five transgenic plant clones that were successfully acclimatized under glasshouse conditions. Among them, BT4 was found to be very slow-growing. The BT3 line showed maximum expression of the HpPKS2 gene which surprisingly also upregulates the expression of the other metabolic pathway genes of B. monierri namely isopentyl- diphosphate delta isomerase (IDDI), squaline synthase (SQS) and acetyl CoA C acetyltransferase (AA). The HPLC analysis in the heterologous system revealed the maximum production of bacopaside I (9.86 +/- 1.0 mg/g dry wt), bacopaside II (5.89 +/- 0.9 mg/g dry wt) and bacopasaponin C (3.59 +/- 0.3 mg/g dry wt) by transgenic lines BT8, BT3 and BT4, respectively. This enhancement in bacopaside I, bacopaside II and bacopasaponin C production was more than 26-fold, 5-fold and 21-fold, respectively in comparison to the control non transformed plants. Key message Overexpression of the HpPKS2 gene in H. perforatum led to higher hypericin content in the native system while its expression in the heterologous system i.e. B. monnieri also improves bacopaside I, bacopaside II and bacopasaponin C production.</p>
10aAgrobacterium tumefacience10aBacopa monnieri10aBacopasides10aHeterologous expression10aHpPKS210aHypericin10aHypericum perforatum10aTransgenics1 aKhan, Shamshad, Ahmad1 aVerma, Priyanka1 aParasharami, Varsha, A.  uhttp://library.ncl.res.in/content/homo-and-heterologous-expression-hppks2-gene-hypericum-perforatum-and-bacopa-monnieri