Floral biology of Mikania micrantha in relation to pollination

BOTA 41066Economic botany and plant breeding

A.T. Wickramage+94716523306

Mikania micrantha

An invasive vineMost 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.

Classification of Mikania micrantha

Kingdom: PlantaeDivision: MagnoliophytaClass: MagnoliopsidaOrder: AsteralesFamily: AsteraceaeGenus: MikaniaSpecies: Mikania micrantha Common names: American rope, Chinese creeper, Mile-a-minute

weed

Seed dispersal

Seeds are dispersed over long distances by wind, animals and by water currents. The germination percentage of seeds is very low (8-12%) compared to other weedy species. Light, water, soil nutrients and fire affect the germination of seeds.

Habitat

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.

Materials

Micania micrantha flowers

Light microscope

Electronic balance

Dissecting microscope

Pipette (10.00 ml), micropipettes

Ruler

Needle

Blade

Watch glasses

Dropper

Slides and cover slips

Test tubes

Distilled water

Sucrose

Methodology

2.2.1 Determination of floral morphology of Mikania micrantha

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.

2.2.2 Determination of the time of the flower opening

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.

2.2.3 Determination of the out crossing index (OCI)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 receptivityThe 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

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

Table 1: OCI values and their corresponding breeding systems

• 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.

2.2.4 Determination of pollen: ovule ratioNumber 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 ovulesCross 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.

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

Table 2: Pollen: ovule ratio and their corresponding breeding systems

2.2.5 Determination of best concentration for Pollen germination

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.

2.2.6 Determination of stigmatic receptivity

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.

2.2.7 Observation of floral visitors

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

3. Results

3.1 Floral morphology

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

Capitulum

Florets

Sepals

Bracts

Style branches

Fused anthers (5)

Petals (5)

Mikania micrantha flower (x 15)

Ovule

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

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

3.2 Floral stages

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

Stage AFloral buds at day 1 (x10)

Stage B Buds are beginning to open at day 2 (x10)

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.

Stage C

Flower is beginning to openat day 3 (x15)

Cross section of stage C (x20)

Anthers

Style

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.

Stage D

Flower just opened at day 4 (x20)

Style is further growing and its lower part bends inside the corolla tube.

At this stage and onwards, insect visitors were observed.

Stage E The style is out of the anther tube at day 5

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.

Anthers

Style branches

Stage FStyle branches separates and curves at day 6 (x25)

Stage G

Style branches expose the stigmatic surface and the anthers are dry, brown in color at day 7 (x25)

Drying anthers

Stigmatic surface

Style branches

• 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.

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

Seeds of Mikania micrantha (x 4)

3.3 Out Crossing Index Character Results

Diameter of the flower or inflorescence (0-1 mm) =1 (1-2 mm) = 2 (2-6 mm) = 3

2

Temporal separation of anther dehiscent and stigma receptivity Homogamy, Protogyny = 0 Protandry = 1

1

Spatial positioning of the stigma and anthers Same level = 0 Spatially separated = 1

1

Total 4

OCI value Breeding System

4 Partially self-compatible, out crossing demand for pollinators

3.4 Pollen: ovule ratioReplicate flowernumber

Number of pollens for 1µl

Number of pollens per flower

Number of ovules

Pollen ovule ratio

1 2 20000 1 200002 3 30000 1 300003 0 - 1 -4 1 10000 1 100005 2 20000 1 200006 1 10000 1 100007 3 30000 1 300008 1 10000 1 100009 1 10000 1 1000010 1 10000 1 10000

• Mean number of ovules per flower = 1

• Mean pollen: ovule ratio = 15000

Pollens through the microscope (10x10x1)

3.5 Determination of best concentration for Pollen germination

Concentration of sucrose solution (%)

Total number of pollens

Number of germinated pollens

Germination 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

3.6 Stigmatic receptivity

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.

3.8 Pollination syndromesThe 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.

3.7 Floral visitors

Ant floral visitors

Bee floral visitors

Butterfly floral visitor

Fly floral visitors

4. Discussion

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).

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.

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.

5. Conclusion

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

6. References 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/mikania-micrantha/images/mikania-pest-fact-sheet-APFISN.pdf (24.06.2013)

http://www.fs.fed.us/global/topic/invasives/feb2007.pdf (24.06.2013)

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