XXIIIrd International Eucarpia symposium, Section Ornamentals ...

OVEREXPRESSION AND SILECING OF
KXHKN5 GENE IN KALANCHOE X
HOUGHTONII
K. x houghtonii (n=51), a triploid interspecific hybrid between K. daigremontiana Hamet
& Perrier (n=17) and K. delagoensis Ecklon & Zeyher (n=34), forms epiphyllous buds on
leaf margin notches in response to a long day photoperiod (vegetative vivipary). Several
well known class 1 knox genes, as Kn1 from maize, STM, KNAT1 and KNAT2 from A.
thaliana and Bkn3 from barley, play an important role in meristem formation and
maintenance and a few reports suggest that this class of homeotic genes could be
involved in vivipary. In attempt to characterize KNOX genes involved in vegetative
vivipary in K. xhoughtonii, overexpression and post transcriptional gene silencing
(PTGS) experiments of KxhKN5 (EU240661) were performed. To accomplish overexpression,
the complete cDNA sequence of the gene (1161 bp), overdrive by 35S
promoter and NOS terminator, was cloned in the binary vector pGreen II
(www.pgreen.ac.uk) that contain the NPTII gene, that confer resistance to kanamycin
(pGreen II NPTII). PTGS construct was prepared by cloning in pJM007 (Schattat et al.,
2004), a 326 bp fragment of the gene in sense and antisense orientation in the specific
cloning sites located at the left and at the right of the PIV2 intron; silencing cassette was
excided from pJM007 and cloned into the binary vector pGreen II NPTII; the derived
vectors were then transferred to A. tumefaciens. Genetic transformation and selection on
medium containing kanamycin, and cefotaxime were conducted; the regenerated shoots
were isolated from the leaf explants and separately cultivated on propagation medium to
establish plant clones some of which were acclimatized in greenhouse. In addition, to
localize KxhKN5 mRNA in early fase of K. Xhoughtonii epiphylly, in situ hybridization
was performed with digoxigenin (DIG)-labelled RNA probe according to DIG RNA
labelling KIT ( Roche, Mannheim, Germany), with minor modifications. Overexpression
and PTGS of KxhKN5 affect plant architecture and leaf shape as reported for class 1
KNOX genes cloned from other species. Ectopic buds and shoots formation was not
observed in transgenic plants overexpressing KxhKN5; furthermore the vegetative
vivipary was reduced in the transgenic clones with extremely modified phenotype likely
as a consequence of the leaf shape modification.
L35
Marina Laura
Cristina Borghi
Cristina Regis
Arianna Cassetti
Andrea Allavena
CRA – Unità di Ricerca per
la Floricoltura e le Specie
Ornamentali, Corso Inglesi
508, 18038 Sanremo (IM),
Italy
a.allavena@istflori.it
Session Molecular breeding 71