IV) RESEARCH AND METHOD

A. PLANT COLLECTION

The E.coccinea , Tuhau plant is collected at Kg.Poring-poring by using cutter ,

while Echinochloa crusss-galli (L.)P.Beauv and Fimbristylis miliacea
(L.)Vahl were collected at KOMSAT , UiTM also by using a cutter .

B. PLANT PREPARATION

The E.coccinea , Tuhau plants is a wild herb from the ginger family. Young,
mature and old leaves were collected . The leaves were washed out several
times with tap water and dried in an oven at 45C for 72 hours. Leaves were
cut into 1 cm pieces, ground into powder in a blender, and sieved through a 40
mesh (420 m) sieve (Phuwiwat et al., 2012).

The racemes from the Fimbristylis miliacea (L.)Vahl seeds were manually
harvested when they had one third of the grains shed and taken to a laboratory
to complete the drying of the seeds under shade conditions where they
remained for four days. After that period, the racemes were gently shaken over
a paper sheet so as to release the seeds (Martins; Martins, 2013). The material
that stayed on the rachis was discarded due to the fact that that material usually
is composed of empty spikelets or immature seeds.

For Echinochloa crusss-galli (L.)P.Beauv , the seed were obtained by mild

shaking of the panicles (Martinkova & Honek, 2013).
C. SAMPLE EXTRACTION

Aqueous extracts were prepared from dried young, mature and old leaves of
E.coccinea , Tuhau by dissolving 10 g of each powdered material in 100 mL of
distilled water at 10C for 72 hours followed by filtration through three layers
of cheesecloth to remove any debris. The supernatant was then filtered through
Whatman No. 1 filter paper to a concentration of 100 g/L of dried plant
material and stored in a refrigerator at 5C until bioassay. Dilutions of
E.coccinea , Tuhau extract (12.5, 25, 50 and 100 g/L) were prepared in distilled
water (Phuwiwat et al., 2012).

D. WEEDS SEED INHIBITION

In order to break the seed dormancy of Fimbristylis miliacea (L.) Vahl seeds
should be carried out under alternate temperatures of 15-25 or 15-35 C in
ready-made substratum or in soil after being dried at a temperature of 50 C for
12 hours. The immersion of seeds in water at environmental or warmed
temperature breaks seed dormancy due to the removal of soluble inhibitors
found in the seed or fruit coat (Brasil, 2009; Lacerda et al., 2010).

For Echinochloa crusss-galli (L.) P. Beauv to break the seed dormancy there
must placed on moistened filter paper at at the temperature of 45 to 50C, but
slightly at 40C ( Miyahara et al., 1974).

E. BIOASSAY

Seeds of chinochloa crusss-galli (L.) P. Beauv were collected from KOMSAT,

UiTM and placed in oven for three days and then incubated in a hot-air oven at
45C for 48 hours to break their dormancy if present. Seeds of Fimbristylis
miliacea (L.) Vahl were collected from KOMSAT, UiTM site. Twenty healthy
seeds of both bioassay plants (which had imbibed for 24 hours in distilled
water)were placed in separate petri dishes (9 cm in diameter) lined with two
sheet of filter paper and moistened with 5 mL of 12.5, 25, 50 and 100 g/L of
each aqueous extract. Four replicates were maintained per treatment for
 each of the two bioassay species in a completely randomized design in a growth
chamber (2532C temperature, a 12/12 hours dark/light photoperiod and 80%
relative humidity). Treatments with distilled water were used only as control.
Germination was deemed to have occurred only after radicle had protruded
beyond the seed coat by at least the dimension of seed at seven days after
treatment. The germination percentage and inhibition rate were measured after
a week (Phuwiwat et al., 2012).

F. DATA ANALYSIS
F(a) INHIBITION RATE
The inhibition rate are calculated based on formula below :

INHIBITION RATE :
|( Control - Extract )| x 100
Control

Figure 1 : Inhibition rate calculation based on Ahn and Chung (2000)

F(b) GERMINATION PARAMETER

Seeds were considered as germinated when their seminal root reached at least 1
mm length. In this study, we used following germination parameters include
Germination percentage (GP, %), Germination rate (GR), Relative germination
percentage (RGP), Mean germination time (MGT), Germination index (GI)
and Weighted germination index (WGI) .
These parameters were also calculated from the formulas proposed by
(Figueroa and Armesto, 2001; Bu et al., 2007; Wu and Du 2007).

GP = 100 GN / SN
GN is the total number of germinated seeds, SN is the total number of seeds
tested
RGP = GP treatment / GP control 100

GI = ((N-i) Gi) 100)/(N GN)

GI is a synthetic measure designed to reflect the synthetical germination ability

including germination rate and germination numbers. Where i is the number of
days since the day of sowing and Gi is the number of seeds germinated on day i.
A weighted germination index (WGI) as described by Bu et al. (2007) was
calculated with maximum weight given to the seeds germinating early and less
to those germinating late .

WGI = [Nn1+(N-1) n2+(N-Z) n3+]/ NN'

where n1, n2, , n60 are the number of seeds that germinated on first, second,
and subsequent days until the 60th day, respectively; N is total days of
experiment; N is the total number of seeds placed in incubation .

GR = Gi / ni Gi

Where i is the number of days since the day of sowing and Gi is the number of
seeds germinated on day i .