Promoter Trapping and Deletion Analysis Show Arabidopsis thaliana APETALA2 Gene Promoter Is Bidirectional and Functions as a Pollen- and Ovule-Specific Promoter in the Reverse Orientation.

The Arabidopsis thaliana promoter trap mutant Bitrap-112 expressing green fluorescent protein (GFP) gene in the ovules was found to carry transferred DNA (T-DNA) insertion at -309 position of the APETALA2 (AP2) gene. Bitrap-112 line did not show phenotype associated with the AP2 mutation, suggesting that T-DNA insertion did not interrupt the AP2 promoter. Further, head-to-head orientation of GFP and AP2 genes indicated that the AP2 promoter could be bidirectional. A detailed deletion analysis of the upstream sequences of the AP2 gene was done to identify the promoter. GUS assay of transgenic A. thaliana plants carrying various AP2 upstream fragments fused to the uidA gene showed that ~200-bp 5' UTR sequences are capable of driving gene expression at low levels in vegetative tissues whereas inclusion of further upstream sequences (~300 bp) enhanced uidA expression comparable to native AP2 expression levels in various tissues including ovules. In the reverse orientation, the 519-bp AP2 upstream fragment was found to drive gene expression in immature ovules and pollen. Absence of antisense transcripts corresponding to the sequences upstream of AP2 gene in wild-type A. thaliana plants suggests that promoter trapping has uncovered a cryptic promoter, which in reverse orientation is capable of driving gene expression in ovules and anthers.

Enhanced shoot multiplication in Ficus religiosa L. in the presence of adenine sulphate, glutamine and phloroglucinol.

Ficus religiosa (Pipal) is a long-lived valuable multipurpose forest tree. The tree is exploited because of its religious, ornamental and medicinal value and the regeneration rate in natural habitat is low. An in vitro propagation protocol has been developed from nodal segments obtained from a 45-50-year old tree. The highest bud break frequency (100 %) followed by maximum number of multiple shoots (13.9) as well as length (2.47 cm) were obtained on Woody Plant medium (WPM) supplemented with 1.0 mg/l BAP along with 0.5 mg/l IAA. Two modifications in this medium resulted in enhanced shoot regeneration-one with 200 mg/l glutamine + 150 mg/l ADS (called as MM-1) giving 32.5 shoots per nodal explant while another modification-with 200 mg/l glutamine + 150 mg/l ADS + 100 mg/l phloroglucinol (called as MM-2) giving 35.65 shoots per explant. These two media were used for sub-culturing of shoots for 4 months. The rate of shoot multiplication was same during the first three sub-cultures on MM-1 and the shoots regenerated were healthy, afterwards shoot multiplication declined. While on MM-2, shoot multiplication declined after first sub-culture and shoots underwent the problem of early leaf fall. Rooting was best induced in micro-shoots excised from proliferated shoot cultures on semi-solid as well as liquid WPM modified with 2.0 mg/l IBA and 0.5 mg/l IAA. The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 25-30 days before transfer to soil conditions, where the established plants showed more than 90 % survival.