Studies of in vitro flowering and de novo flowers of Nicotiana tabacum

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Virginia Polytechnic Institute and State University


The objectives of this research were to examine factors influencing de novo flowering of Nicotiana on 2-3 x 10mm explants consisting of epidermal and 3-6 layers of subjacent cells (thin cell layers, TCLs) and to compare de novo to in vivo flowers.

TCLs from short-day and long-day tobacco plants were compared with TCLs from day-neutral species to examine in vitro floral photoinduction and graft transmissibility of floral promoters and inhibitors. TCLs from photoperiodic species of tobacco did not form flowers de novo , whereas TCLs from day-neutral plants did flower. When TCLs were removed from photoperiodic plants and grafted in vitro to TCLs from day-neutral plants, there was no indication that a floral-promoter or inhibitor was transported through the non-vascular graft union. In vitro photoinduction of TCLs removed from photoperiodic plants was not possible under conditions conducive to in vitro flowering of TCLs from day-neutral species.

TCLs taken from intraspecific F₁ and F₂ hybrids between short-day and day-neutral cultivars of N. tabacum were examined to assess the importance of genotype and photoperiod to de novo flowering. Flowering of the F₂ population occurred over a 9 week period under naturally decreasing photoperiod. Photoperiodic response and in vitro flowering were correlated in the F₂ population with fewer flowers produced per TCL with increasing short-day reaction. F₂ segregates whose TCLs did not yield de novo flowers were found among both day-neutral and short-day phenotypes.

When de nova flowers were compared to in vivo flowers of diploid (2n=4x=48) N. tabacum 'Samsun' and haploid (2n=2x=24) plants derived from 'Samsun' anther culture, major morphological differences were found. Flower and anther sizes were reduced in de novo flowers and the numbers of anthers and pistils produced per flower were variable. TCLs from haploid plants produced more flowers in a shorter period of time than TCLs from diploid plants. Anthers cultured from de novo haploid plants were embryogenetic resulting in mixoploid plants; anthers from in vivo haploid flowers were not embryogenetic. Anthers from in vivo diploid plants were five times more embryogenetic than anthers from either de novo haploid or diploid flowers. Meiotic analysis revealed similar abnormalities from both in vivo and de novo microsporogenesis of haploids.