1. BOTA 41066
Economic botany and plant breeding
A.T. Wickramage
+94716523306

2. An invasive vine
Most commonly called as the mile-a-minute
plant.
It has its origins in the South American
rainforests, where it grows near rivers and in
disturbed areas near forests.
Introduced to South Asia originally to
camouflage airfields during the World War II.

3. Kingdom:
Plantae
Division:
Magnoliophyta
Class:
Magnoliopsida
Order:
Asterales
Family:
Asteraceae
Genus:
Mikania
Species:
Mikania micrantha
Common names: American rope, Chinese creeper, Mile-a-minute
weed

4. Seeds are dispersed over long distances by
wind, animals and by water currents. The
germination percentage of seeds is very low (812%) compared to other weedy species. Light,
water, soil nutrients and fire affect the
germination of seeds.

5. Wet places, forest borders and clearings, along
the banks of streams an drivers, roadsides and
railway tracks, in pastures, forest plantations,
agricultural and agro - forestry systems, open
disturbed areas and barren lands. Mikania
grows luxuriantly on leached and nutrient poor
sandy loam to clayey soils. The weed cannot
tolerate shade and hence fails to penetrate
undisturbed natural forest areas.

6. Micania micrantha flowers
Needle
Light microscope
Blade
Electronic balance
Dissecting microscope
Watch glasses
Dropper
Slides and cover slips
Pipette (10.00 ml), micropipettes
Test tubes
Ruler
Distilled water
Sucrose

8. Selected Mikania micrantha buds were labeled
with tags.
Morphological characters of the Mikania
micrantha flowers were observed; color,
diameter, ovary position, number of ovules,
number of petals, number of sepals, number of
stamens, number of styles of the flower were
determined.

9. A flower bud was selected and it was covered
and tagged without harming the structures.
The flower bud opening and orientation
changes were observed daily and the flower
opening stages were photographed.

10. Three characters consider for this. They are,
1. Diameter of the flower
Diameter of the flower was measured using a ruler. Five replicates were done.
[Diameter of the flower or inflorescence (0-1 mm) = 1, (1-2 mm) = 2, (2-6 mm) = 3]
2. Temporal separation of anther dehissecence and stigma receptivity
The excised pistil was immersed in a watch glass that contains hydrogen peroxide.
Bubble formation was observed.
External observation of stigma was also used.
(Homogamy, protogyny = 0 potandry = 1)
3. Spatial position of the stigma and the anther
Spatial position of the stigma and the anther was determined by observing externally.
Same level = 0
Spatially separated = 1

11. • Floral character was ranked according to the out cross index.
• Sum of the out crossing values were taken and by that their breeding system were
determined.
Table 1: OCI values and their corresponding breeding systems
OCI value
Breeding System
0
Cleistogamy
1
Obligate autogamy
2
Facultative autogamy
3
Self compatible, some demand for pollinators
4
Partially self compatible, out crossing demand for
pollinators

12. Number of pollen grains
Pollens were collected before anthesis by crushing anthers of a flower using a
slide, then the macerate was put in to a test tube with 10.00 ml distilled
water. And drop of cotton blue was added. The suspension was well mixed.
A volume of 1µl was added on a microscopic slide, and covered with a cover
slip. Pollen grain number was counted. Then the value was calculated for one
anther. The observations were done under the light microscope 10 x 40
power.
Number of ovules
Cross sections of ovary were taken and ovules were counted under dissecting
microscope.
Number of pollens and ovules were counted in 10 replicates. Mean number
of ovules and pollens were calculated. Breeding system was determined
using these data of pollen: ovule ratio.

13. Table 2: Pollen: ovule ratio and their corresponding breeding systems
Pollen / Ovule ratio
Breeding System
2.7 - 5.4
Cleistogamy – without pollination
18.1- 39
Obligate autogamy - Self pollination
31.9 - 396
Facultative autogamy
244.7 - 2588
Facultative xenogamy
2108 - 195525
Obligate xenogamy – Cross pollination

14. Percentages of 0, 5, 10, 15, 20 and 25 of sucrose concentration series were
prepared. Pollen grains from one anther were added separately to the each
solution. Five cavity slides were taken and were labeled according to the
above sucrose concentrations. One drop of each solution was taken on cavity
slide. The cavity slides were kept in a moist chamber for 30 minutes. The
slides were observed under microscope at different time intervals and
observed the germinated and non-germinated pollens. The best
concentration for the germination was determined by calculating
germination percentages at each concentration. The pollens were taken at
different time of the day and the above procedure was followed with best
concentration of sucrose solution.

15. From each flowering stages, flowers were taken
and H2O2(aq) 6% solution was added to their
stigmatic area with a dropper. Any sign for air
bubbling/ air bubbling rate was observed in
each flower.

16. Flower visitors were observed during the time
of flower is in open.

18. Character
Observation
Color of petals
White
Diameter
1.5 mm
Number of ovules
1
Ovary position
Inferior
Number of petals
5
Number of sepals
5
Number of stamens
5
Number of styles of the flower
1

19. Capitulum
Florets
Sepals
Bracts

20. Style branches
Fused anthers (5)
Petals (5)
Mikania micrantha flower (x 15)

21. Ovule
L.S of Mikania micrantha flower
ovary (10x10x1)
C.S of Mikania micrantha
flower ovary (10x10x1)

22. The flowering period is 8 days
Can be divided into eight floral stages (A, B, C,
D, E, F, G and H) based on style morphology and
behavior.

23. Stage A
Stage B
Floral buds at day 1 (x10)
Buds are beginning to open at day 2 (x10)

24. Stage C
Flower is beginning to open
at day 3 (x15)
A growing filament raises the anther tube up to the same level as the
style
The style branches are aligned and the stigma is not yet receptive.
The flower opens and the tip part.

25. Anthers
Style
Cross section of stage C (x20)

26. Stage D
Flower just opened at day 4 (x20)
The style breaks through the anther tube formed by five
fused anthers, anthesis happens.
The style begins to protrude out of the anther tube and the
pollen grains are removed from the anthers by the
sweeping hairs of style.

27. Stage E
The style is out of the anther tube at day 5
Style is further growing and its lower part bends inside the
corolla tube.
At this stage and onwards, insect visitors were observed.

28. Style branches
Anthers
Stage F
Style branches separates and curves
at day 6 (x25)
The growing style unthreads through the tube and
loosens the pollen grains which adhere to the sweeping
hairs of the style branches.
As the stage continues the style grows longer from the
anther tube and presented more pollen attached on the
sweeping hairs of the style.
At this stage floret was completely open. The style and
style branches appear in yellow color.

29. Style branches
Stigmatic surface
Stage G
Drying anthers
Style branches expose the stigmatic surface
and the anthers are dry, brown in color at day 7 (x25)
• The style grows to its full length, its two branches completely open.
• Anthers become dry and brown and begin to wither
• Scent emission still continues.

30. Stage H
Senescence stage of the floret.
The two style branches bending towards the center
of the floret at day 8 (x20)
• Flowers enter the senescence stage
• The two style branches bend towards the center of the floret

31. Seeds of Mikania micrantha (x 4)

32. Character
Results
Diameter of the flower or inflorescence
2
(0-1 mm) =1
(1-2 mm) = 2
(2-6 mm) = 3
Temporal separation of anther dehiscent and stigma receptivity
1
Homogamy, Protogyny = 0
Protandry = 1
Spatial positioning of the stigma and anthers
1
Same level = 0
Spatially separated = 1
Total
4
OCI value
Breeding System
4
Partially self-compatible, out
crossing demand for pollinators

33. Replicate
flower
number
Number of
pollens for
1µl
Number of
pollens per
flower
Number of
ovules
Pollen ovule
ratio
1
2
3
4
5
6
7
2
3
0
1
2
1
3
20000
30000
10000
20000
10000
30000
1
1
1
1
1
1
1
20000
30000
10000
20000
10000
30000
8
9
10
1
1
1
10000
10000
10000
1
1
1
10000
10000
10000
• Mean number of ovules per flower = 1
• Mean pollen: ovule ratio = 15000

34. Pollens through the microscope (10x10x1)

35. Concentration of
sucrose solution (%)
Total number of
pollens
Number of germinated Germination
pollens
percentage
0
4
0
0%
5
6
0
0%
10
5
1
20%
15
3
1
33%
20
2
0
0%
25
4
0
0%
The graph of germination vs. concentration of sucrose solution

36. Stage F and G stigmas gave a higher rate of
bubbling with H2O2.
At these stages stigmatic surfaces were white in
color and they had a glistening, wet
appearance. Also at these stages anthers were
dry and brown in color.

37. The flowers open at any time of day.
Nectar is present.
Flowers are slightly fragrant. Higher fragrant level can be
observed at initial stages (A- G) of flower and minimum at the
senescent stage (H).
Most of the pollinators present at stage E and F.
Floral visitors can be mostly seen at 11.00 am to 3.00 pm, when
there is high sunlight.

38. Bee floral visitors
Ant floral visitors

39. Fly floral visitors
Butterfly floral visitor

40. M. micrantha is a protandry flower. Anthesis happens before the
stigma become receptive/ become mature. Stigmatic receptivity
can be identified by the higher bubbling rate with H2O2, which
was observed at stage F and G.
Also stigma and anthers in different levels, the stigma is in
higher level than anthers. This spatial arrangement of the stigma
and anthers reduces the possibility of contamination by self
pollens (if there are any viable pollens when the stigma is
receptive) and thereby further facilitate out crossing.
By approach herkogamy (pin) arrangement of stigma and
anthers causes floral visitors to first contact the stigma, before
removing pollen from the anthers (if there are any viable pollens
left).

41. Two style branches act as pollen presenters which are
responsible for removing pollen out of the anther tube while the
terminal section of the style branches grows through anthers
and presenting/exposing pollens to the floral visitors/
pollinators. Also the bending behaviour of the M. micrantha
style branches help the growing style to move the pollens out of
the anthers efficiently. This happens when the style branches
are joined and the stigmatic surfaces are not receptive also
when the pollen has the highest viability. This mechanism is
known as secondary pollen presentation. These morphological
and structural characteristics of the style branches and pollen
are adapted to facilitate cross pollination.
However self-pollination cannot be totally excluded. Receptive
stigma is temporally separated from the floret’s own viable
pollen, but cannot completely avoid the probability of autogamy
or geitonogamy.

42. Even though, the pollen counting through the microscope is
much easier technique; there may be mistakes and errors in
handling of anthers, using anthers before the dehiscence,
counting. In the case of M. micrantha it is important to identify
the correct male stage of flower before the anthesis and careful
dissecting.
In determination of best concentration for pollen germination
using sucrose concentration series, only 10 and 15 percent
sucrose solutions show germination. This lack of germination in
other concentrations may due to selection of floral stages of not
viable pollens.
The common floral visitors are ants, bees, butterflies, other flies.
The flowers have white colored petals, nectar, and scent to
attract pollinators.

43. Mikania micrantha flower is an obligate
xenagamous flower- cross pollination, and it
demands for pollinators.

44. Hong, L. Shen, H. Ye, W. Cao, H. and Wang, Z. Secondary pollen
presentation and style morphology in the invasive weed Mikania
micrantha in South China. Botanical Studies (2008) 49: 253-260.
http://www.publish.csiro.au/paper/BT9930417.htm
(24.06.2013)
http://www.freshfromflorida.com/pi/mikaniamicrantha/images/mikania-pest-fact-sheet-APFISN.pdf
(24.06.2013)
http://www.fs.fed.us/global/topic/invasives/feb2007.pdf
(24.06.2013)