Department of Biological Sciences

College of Art and Sciences

Visayas State University
Visca, Baybay City, Leyte 6521-A, Philippines

Name: Reina L. Cresino BS-Biology 3 Date Submitted: March 2, 2018

Group no. 5 Subject: Molecular biology

Exercise no. 2
Cellular Changes Associated with Development

Introduction

Hibiscus L. is a genus of the family Malvaceae, and contains about 300 species mainly growing
in tropical and subtropical areas. Plants from the genus grow as small trees, shrubs and herbs. H. rosa-
sinensis (Hawaii flower, Rosa of China or shoe flower) is one of the most widely planted ornamental shrub
throughout the tropics and is an important potted plant in Europe and USA. The greatest diversity of the
species is found in India, China, South-East Asia and South-Indian Ocean Islands. Hibiscus rosa-sinensis has
limitation of its use as an outdoor ornamental plant in a temperate climate is due to chilling sensitivity.
Cold hardiness is a highly complex trait, involving multiple genes. Improvement of H. rosa-sinensis for
increased chilling tolerance by conventional breeding is unlikely because there is a lack of genetic
information in relation to this trait within this species. In the Hibiscus genus, cold hardiness is available,
but only in a few species. Attempts to create cold tolerant Hibiscus plants resembling H. rosa-sinensis
suitable for Nordic climate have not been successful so far. Pollen viability was determined by staining
with Cotton blue. An attempt to resolve the ability of pollen grains to germinate was made in vitro using
artificial medium.

The potential yield of viable pollen collected from the field is influenced by season and timing
of flowering, quantity of pollen set, and method of handling and storing pollen. There are many particular
factors that determine pollen availability, such as genetic controls, temperature and moisture, collecting
time, radiation, chemicals applied to plants for insect control, and industrial waste gasses. Low or high
temperatures during the development period can adversely affect the quantity and germination response
of mature pollen. Pollen isolated in early morning germinates better than that collected at other times of
the day. This is probably related to metabolic transitions and moisture stresses. Many factors also affect
the viability of pollen in storage. The amount and quality of pollen produced by a flower are important
components of fitness. The quality of pollen is assessed on the basis of viability and vigor of the pollen
grain (the proportion of pollen grains that are viable). Pollen vigour refers to the speed of germination of
pollen grains and rate of pollen tube growth. There are four phases in pollen germination and tube growth:
imbibition phase, lag phase, tube initiation phase and rapid tube elongation phase. Pollen of many species
can be stored at temperatures between 4°C and -20°C for the short term. The life span of pollen grains
has been reported both in in vitro and in vivo germination tests as lasting for a few minutes up to several
days. The viability of pollen has been investigated in terms of its contribution to compatibility and fertility
for crop improvement purposes. Several studies also assessed pollen viability by staining and direct count.
Commonly, in vitro pollen germination tests have been used to determine pollen germination percentage
and can be used for assessing pollen vigour by monitoring germination rate over a period of time, or by
measuring the length of pollen tube formation. Most pollen viability tests germinate small samples of
pollen and observed under microscope to count grains percentage of producing tubes over time. Several
replicates may be tested and germination percentage is taken as an index of pollen viability. The main
problem with in vitro germinations to find suitable testing medium for each cultivar. Many pollen grains
can germinate in water or aqueous solutions of sucrose with no additives, but pollen types such as tri-
nucleated grains need special substrates for germination because tri-nucleated grains have abnormally
short pollen tubes and reduced to protuberances. Sucrose is the best carbohydrate source for pollen
germination and tube growth for most plants. Under natural conditions, pollen of different species often
require specific media for germination. The liquid medium is usually used in current protocols for pollen
germination. Research on pollen germination using different media has been carried out on a wider range
species.

In vitro germination tests have often been performed to evaluate the duration of pollen viability
and the ability of aged pollen to produce fruits and seeds containing embryos; also to determine the
effectiveness of pollen storage procedures for breeding programs and genetic conservation.

Objectives

 To study cellular changes associated with development.

 To relate cellular changes with cell function.
 To prepare mounts and interpretative drawings/photomicrographs.
 To apply some techniques in cell preparation for microscopic observation.

Methodology

A. In pollen grain staining as test for pollen viability.

First the pollen grain is collected into the surface of the glass slide and it was been putted a stained or it
was stained using Cotton blue then carefully putted the cover slip to avoid bubbles to appear, After the
stain was already penetrated in the cell wall of the cell it is been viewed in the microscope in LPO.

B. In vivo Pollen germination and growth.

In vivo pollen germination and growth we collected a flower buds that are about to open or the mature
buds the tip of it were cut and it was lightly putted a pollen grain in the stigma. 30 minutes is the interval
of each so each slide had 30 minutes, the second is 60 minutes, third is 90 minutes and lastly is 120
minutes. After its designated time the style were cut and stained with cotton blue then it is cover with
the glass slide. It is the same as will the other sample will undergo.

C. In vitro Pollen Germination and Growth

In In vitro Pollination and growth, a petri dish were lined with absorbent paper then it was, or the sample
in the glass slides were putted a 10% sucrose solution, to distribute it well, a forceps were used to scatter
all the pollen grain into the glass slide then it was incubated for 30, 60, 90, 120 minutes for each slide.
After a 30 minutes the slide was being viewed in the microscope to see what happened during the 30
minutes lapses and as will as the other were viewed after their assigned minutes that they were assigned
to view on that time then it is observed each result.

Discussion

Pollen viability, as estimated by staining with Cotton blue. The percentage of fertile pollen was
75-80% for H. rosa-sinensis which is has also have a not viable pollen grains present and is also estimated
that there were 5% that are not viable. When we say viable it is the capacity to live or to succeed in
pollination or in life. There are many factor’s that been stated above that may affect the fertile or the not
viable pollen grain it can be caused by the factor like temperature, weather, climate or etc. In in vivo the
H. rosa-sinensis or the buds of it were collected for the test of germination and growth the results are
shown below in figure 1, 2, 3, and 4.

Figure 1: Result after 30 minutes Figure 2: Result after 60 minutes

Total Magnification: 100X Total Magnification: 100x

As we can see above the results, in the figure 1 which is the result after 30 minutes it is more clearly seen
above that there were no pollen tube were present or visible at all same as the figure 2, the result after
the 60 minutes of wait and same in the result after the 30 minutes there were also no pollen tube that is
been present in the stigma. Maybe it do more need more time to develop its pollen tube and in below
this are result after 90 minutes and 120 minutes.
 Figure 3: Result after 90 Minutes Figure 4: Result after 120 minutes

Total Magnification: 100X Total Magnification: 100X

As we can also see result the result above in figure 3 and Figure 4, in figure 3 there is small growth of the
pollen tube present in the stigma while in the figure 4, where 2 hours had been lapse there were obvious
presence of the pollen tubes. In the below these are the result of the in vitro which the pollen grains were
been develop in 30, 60, 90, 120. Shown in the figures below:

Figure 5: in vitro, Result after 30 minutes Figure 6: in vitro, Result after 60 minutes

Total Magnification: 100X Total Magnification: 100X

Figure 7: in vitro, result after 90 minutes Figure 8: in vitro, Result after 120 minutes

Total Magnification: 100X Total Magnification: 100x

As we clearly see the result above in figure’s 7 and 8 after 90 minutes and 120 minutes after. In figure 7
result is it is clearly seen the presence of the pollen tube that been developed after 90 minutes of waiting
and after 120 minutes in figure 8 there is no really doubt that those in above (Illustration/Picture above)
is showing the growth of the pollen tube it is much longer comparing to the result of 90 minutes after in
figure 7. So those are the overall results stated above after conducting the whole experiment.

Conclusion:

Therefore, it was successfully observe the pollen tube present in the Hibiscus rosa-sinensis in 90 minutes
after and much longer in after 120 minutes so, time is a big factor to observe the pollen tube germination
the longer the time it is going to be incubated the more possibilities of being able to see the successful
result of the conducted experiment.

Recommendations:

It should have more time to conduct the experiment and it should have more replicates in each
experiment that is conducted for it to have more possibilities of successful observing the pollen
germination.

Literature Cited:

Akpan, G.A. 2007. HIBISCUS: Hibiscus rosa-sinensis p.479-490 In: N.O. Anderson (ed.), Flower Breeding
and Genetics: Issues, Challenges and Opportunities for the 21st Century. Springer, Dordrecht.

Gandadikusumah VG, Wawangningrum H, Rahayu S (2017) Pollen Viability of Aeschynanthus tricolor

Hook. J. Trop. Life. Science 7 (1): 53 – 60.