D E N D R O B I U M O R C H I D S C A R RY I N G A N T I S E N S E A C C Ni Putu Ayu Erninda Oktaviani

OXIDASE: Suputri
S M A L L C H A N G E S I N F L O W E R M O R P H O L O G Y A N D A D E L AY
O F B U D A B O RT I O N , F L O W E R S E N E S C E N C E , A N D
ABSCISSION OF FLOWERS 21/486694/PBI/1780
 Background

Antisense to delay of
bud abortion, flower
senescence, and
abscission of flowers

Using antisense copies of a Carica

Ethylene (ET) is a plant hormone produced by changing, 1- papaya gene encoding 1-
aminocyclopropane-1-carboxylic acid (ACC) to ET by an enzyme aminocyclopropane-1-carboxylic acid
ACC oxidase (ACO) and influence bud abortion, flower senescence, oxidase (CpACO)
and abscission of flowers
METHOD The 4 transgenic orchid lines (AE1, AE2,
AE3 andAE4) were analyzed for the copy
The construction of binary vector harboring number of antisense CpACO1
antisense CpACO1 gene

Antisense ACO1 gene was cloned from cDNA of Protocorm like bodies
Carica papaya (CpACO1)(Accession No. AY601704) using the primers (PLBs) transformation
aCpACO1F:5’-GGAGCTCGCTAGCCACCATGATCTCTCATGACCTGATGGA-
3’ and aCpACO1R: 5’-GGTCTAGACCATGGTCTACCAGAGAGGTGCTGG-3’.
Antisense CpACO1-
was inserted into pCAMBIA 1301 vector (CSIRO, Australia) to yield
pCAMBIA1301aACO1 plasmid that contains hygromycin phosphotransferase
gene
(hpt) as a selectable marker under the control of CaMV35S promoter.

CpACO1
RESULT AND
DISSCUSION
ACC Concentrations, ACO Activity, And
ET Production

In the inflorescences no difference was

observed between the non-transgenic plants and
transgenic line AE1 (one antisense ACO copy; Fig.
S1). A lower ACO activity than in the controls was
found in the other transgenic lines (Fig. 1). The
decrease of ACO activity in the four lines was
positively correlated with the copy number of
theantisense ACO gene (R2 = 0.96).

The rate of ET production in whole vegetative plants

(i.e. containing no inflorescences) was determined. It
was higher in non-transgenic plants than in
the transgenic lines (Fig. 2a). The ET production of
the transgenic lines AE2-4 was lower than that of
AE1
(Fig. 2a).

The increase in ET production by individual floral

buds attached to the plant, which is related to bud
opening, was delayed in the lines AE2 and AE4 and
delayed and attenuated in line AE3 (Fig. 2b). The
increase in ET production by individual open flowers
attached to the plant, which is related to senescence
of the petals and sepals, was delayed in line AE1, and
more delayed and attenuated in lines AE2, AE3, and
AE4 (Fig. 2c).
Flower senescence on cut Dendrobium inflorescences is preceded by a large increase in ET production
(Ketsa and Prayurawong 2001), and can be delayed by using inhibitors of ET perception or ET
synthesis (Ketsa and Prayurawong 2001; Ketsa and van Doorn 2009; Uthaichay et al. 2007), showing a
role of endogenous ET. The present data suggest that ACO can limit the increase in flower ET production
and the related senescence symptoms. The time to senescence in the (unpollinated) flowers on the plant
was about 20 days in the controls and 21, 24, 25, and 23 days in the transgenic lines AE1–4, respectively
T I M E T O

Interestingly, some aspects of generative development were delayed in intact Dendrobium ACO antisense plants: the time
to yellowing and water soaking of the floral buds and the abscission of these buds. Floral bud abortion can have various
causes. In dendrobium it was due to low light intensity and/or a short photoperiod and could be induced by ET and
prevented by an inhibitor of ET perception. Another process due to adverse water relations it was prevented when the trees
were adequately chilled during dormancy, while no effect of water relations were found. The environmental causes of floral
bud abortion in Dendrobium, if any, are apparently not known. Abortion of generative tissues is a typical response to the
stress induced by lack of resources. The present datashow a role of ET in the process.