Evaluation of Pollen Tube Growth from Liliaceae Flower

Evaluation of Pollen Tube Growth Using Lilium Lancifolium Flower

Sea Coast Science Inc.

Harika Sokkam August 6th, 2014

Committee Members: Dr. Geoffrey C. Landis, Senior Scientist Seacoast Science, Inc. Dr. Sajith Jayasinghe Associate Professor, CSU San Marcos. Dr. Betsy Read, Professor, CSU San Marcos

Professional Science Masters in Biotechnology

California State University San Marcos 333 S Twin Oaks Valley Rd San Marcos, CA-92078

Table of Contents

EXECUTIVE SUMMARY ...... III ACKNOWLEDGEMENTS ...... IV INTRODUCTION...... 2 MATERIALS AND METHODS ...... 6 POLLEN COLLECTION ...... 6 METHOD 1: POLLEN AND STIGMA ...... 8 METHOD 2: POLLEN, STIGMA WITH PART OF STYLE AND OVULES ...... 10 METHOD 3: POLLEN WITH WHOLE PISTIL ...... 12 METHOD 4: POLLEN AND OVULES ...... 13 METHOD 5: JOHNSON-BROUSSEAU AND MCCORMICK METHOD ...... 13 METHOD 6: YONGXIAN LU METHOD ...... 15 RESULTS AND DISCUSSION ...... 17 METHOD 1: POLLEN AND STIGMA ...... 17 METHODS 2-6 ...... 17 CONCLUSION ...... 20 REFERENCES ...... 21

LIST OF FIGURES

Figure 1 Pollen Growth Cell Kit seen under microscope [13] ...... 3

Figure 2 Picture of Lilium lancifolium[11]...... 4

Figure 3 Cross section of Lilium lancifolium flower[12] ...... 4

Figure 4 Reproduction of a Flowering Plant [10] (Encyclopedia Britannica) ...... 5

Figure 5 Pollen being collected from Anthers ...... 6

Figure 6 A mixture of fertilizer solution with dilute detergent in a 5ml container ...... 7

Figure 7 Live pollen of Lilium lancifolium in solution under the microscope ...... 7

Figure 8 Stigma being cut (top) and stigma pieces with pollen on them (bottom) ...... 9

Figure 9 Stigma with pollen attached on top of it in wells with fertilizer solution ...... 10

Figure 10 Ovules of Lilium lancifolium being extracted from ovaries onto a micro slide ...... 10

Figure 11 Stigma, pollen and eggs in wells ...... 11

Figure 12 Stigma, pollen and ovules in wells with more room ...... 12

Figure 13 Whole pistils with pollen in a petri dish with fertilizer solution and agarose gel ...... 12

Figure 14 Johnson-Brousseau and McCormick Method to grow Arabidopsis Thaliana pollen tubes [9] ...... 14

Figure 15 Polycarbonate nucleopore membrane filter pads (top) and agarose gel pads (bottom) used with Lilium lancifolium pollen ...... 15

Figure 16 Stigma and style, with Aniline Blue staining, under a microscope to get background control and stain any other structures...... 16

Figure 17 Dense pollen tube growth, as seen through a microscope (individual pollen tubes are not identifiable) ...... 17

EXECUTIVE SUMMARY

Evaluation of Pollen Tube Growth from Lilium lancifolium Flower

Harika Sokkam

Aug 6th, 2014

Professional Science Masters

California State University, San Marcos

The growth of pollen tubes in the fertilization process of flowering plants is a common area of research. It is also a part of the science curriculum taught in schools. The project’s goal is to study different methods of growing pollen tubes in the lab. Several experiments are conducted using multiple techniques on Lilium lancifolium (Tiger Lily) flowers. Staining techniques are also studied to find an easy way of visualizing pollen tube growth in classroom settings.

The final outcome is that there is dense growth of pollen tubes though it was not possible to isolate individual germinating pollen tube. This is probably due to many difficulties in maintaining the proper conditions for pollen growth in a synthetic media in-vitro outside of the flower. It was also realized that cheaper and easier staining techniques for use as educational aids do not work. The traditional, more expensive staining techniques with appropriate microscopy are required.

iii

ACKNOWLEDGEMENTS

I take this opportunity to express my gratitude to my instructor Geoffrey C. Landis for his support and encouragement throughout the course of the thesis. I learnt a lot from his expertise and professionalism. Completion of the project wouldn’t have been possible without his constant guidance.

I would like to thank my committee members Dr. Betsy Read and Dr. Sajith Jayasinghe for their help and guidance, which helped me through the various stages of the project.

I would also like to thank my husband, Ramesh for his encouragement and support.

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

INTRODUCTION

Seacoast Science Inc. is a company focusing on the chemical sensor and chemical detection market. Their products include micro machined devices to detect vapors of organic and inorganic gases. These devices are compact and low cost.

A new product in development at the company is the Pollen Growth Cell (PGC) Fertilization Kit. This is a transparent micro device designed to support pollen tube growth in a very controlled environment. The device has many potential applications both as an educational tool in schools and colleges as well as in scientific research requiring accurate control of the pollen tube growth environment and measurement of the speed and directionality of the pollen tubes. Some previous research using this device has been conducted on the model research plant Arabidopsis thaliana [1].

Pollen tube growth is a rapid process and the rate of growth is quite fast that it can be measured under a microscope within the time of a class in school to educate students. It is wonderful to see the growth of pollen tubes when they germinate and move from the anther to the stigma and ultimately reaches the ovule at the base of the pistil of the flower.

Pollen is studied because of the fact it causes allergy and hay fever which is interesting to know about allergic reactions to different pollens. Researchers typically perform pollen tube guidance bioassays in an isotropic-ally diffusive environment [1]. This is considered a semi in-vivo assay that occurs inside a Petri dish. It is difficult to perform quantitative analysis of pollen tube growth as it is disorderly in conventional agarose gel medium [3]. The PGC kit (Fig 1) and the plate assay have similar success rates in targeting unfertilized ovules. However, with the PGC kit, the procedures are more robust and consistent in achieving fertilization with higher efficiency [1]. Unlike the plate assay, the PGC kit is capable of creating micro-gradients of guidance cues. This feature allows more sophisticated experiments to be performed.

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

Figure 1 Pollen Growth Cell Kit seen under microscope [13]

Previous research has been conducted on the PGC kit for Arabidopsis thaliana [1]. Other researchers have recently introduced a microfluidic system using similar principles to which can study individual pollen tubes [4]. A wealth of research has been conducted on the pollen growth guidance signals using traditional methods [5, 6].

The project aims to study and compare various methods for pollen tube growth for a commercial plant species, Lilium lancifolium (Tiger Lily). The goal is to find the optimal way of fertilizing tiger lily and test the usage of the PGC kit. After pollen tubes start growing from the pollen, they will be placed in a PGC kit to test the usefulness of the device. We chose not to use A. thaliana in spite of considerable number of previous studies because the pollen, stigma and style are very small, whereas for Lilium lancifolium they are much larger. The large parts are more suitable for a classroom setting, which is the target market for the PGC kit.

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

Figure 2 Picture of Lilium lancifolium [11]

Figure 3 Cross section of Lilium lancifolium flower[12]

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

Figure 4 Reproduction of a Flowering Plant [10] (Encyclopedia Britannica)

Reproduction of flowering plants depends on delivery of the pollen from the anther to the ovule in the stigma, which occurs through a long projection of a pollen cell(s), called a pollen tube. The pollen tube grows only at its tip, and this growth is distinguished by very fast rates, up to 1cm/Hr. and reaches extended lengths of up to 1foot. One of the most common areas of research in pollen biology is the study of how cell wall material can be produced at such fast rates, and how the cell wall deposition and structure are regulated to allow for rapid changes in the direction of growth [2].

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

MATERIALS AND METHODS

Pollen Collection

The first task in all the fertilization methods is to collect and store the pollen. The tiger lily flowers were bought from the florist and the stigma and stamens were physically removed from the flower. Five to six anthers were collected from each anther of Lilium lancifolium flower from which the pollen grains were collected. This was done under a microscope using a bent needle as seen in the Figure 5 below.

Bent Needle

Anthers

Pollen

Figure 5 Pollen being collected from Anthers

Pollen was collected from the anthers and dried for some time and transferred to 5ml glass containers containing 2 ml of fertilizer solution and a few drops of diluted detergent. Pollen in solution is the best way to deliver pollen. The pollen was mixed in these solutions and the fertilizer solution helps for the individual pollen grains to germinate and the detergent solution helps to prevent the pollen from becoming sticky. It is easier to handle and deliver pollen to the tip of the stigma if it is in a solution rather than dry. The fertilizer solution is Miracle Grow, Bloom booster flower food and was prepared by adding 1.89gms of it in 500ml of distilled water.

6 Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

Figure 6 A mixture of fertilizer solution with dilute detergent in a 5ml container

Before using the pollen in the experiments, the viability of the pollen was tested by observing the shape and color of the pollen using a microscope. The pollen is delivered on top of 0.5% agarose gel and observed under the light microscope. Live pollen is bigger, rounded and opaque while dead pollen is smaller, shriveled and clear. The size of the pollen is extremely small and very difficult to work with.

Pollen grain 1 µm in size

Figure 7 Live pollen of Lilium lancifolium in solution under the microscope

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Method 1: Pollen and Stigma

Each tip of the stigma was cut into 3 pieces and they were placed in the 0.5% agarose gel impregnated with the fertilizer solution in each of the wells of a 12 well micro titer plate.

The pollen was delivered on top of the three pieces of stigma.

Whole tip of the stigma

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Part of the stigma with pollen

Figure 8 Stigma being cut (top) and stigma pieces with pollen on them (bottom)

The stigma and pollen were transferred to a cylindrical well of a 12 well microtiter plate. Each well contains 0.5% solidified agarose gel dissolved in the fertilizer solution which is Miracle Grow. On top of each stigma tip, the pollen grain was placed. The fertilizer solution was added to the agarose gel to hydrate the pollen and stigma. This tray was covered with the lid and placed at room temperature for 24 hours as seen in Figure 9. After 24 hours, the agarose gel from each well was removed carefully and cut into thin slices. The slice containing the pollen and stigma was transferred into a 5ml glass container into which 2ml of a dye named 2, 4, 6 trinitrobenzene sulfonic acid was added and incubated for 1 hour. The sliced gel in the container was washed with distilled water four times and is transferred to a glass slide and observed under light microscope. The dye is expected to stain the specific polymer elements in the pollen tubes and be visible under the microscope.

9 Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

Stigma with Pollen

Figure 9 Stigma with pollen attached on top of it in wells with fertilizer solution

The experiment was repeated four times and the wells were observed for pollen tube growth under the microscope.

Method 2: Pollen, Stigma with part of style and Ovules

The second method involved the isolation of ovules of tiger lily under the microscope by dissecting the ovary which is the base of the stigma. The ovules were collected from the ovary and placed on a microslide. The ovules were hydrated with distilled water and kept aside.

Scalpel

Ovules on a microslide

Figure 10 Ovules of Lilium lancifolium being extracted from ovaries onto a micro slide

Two variations of the experiment were conducted.

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

In the top well of Figure11, two stigmas along with a small part of style were cut into six pieces and placed in a petri dish with some drops of water to hydrate them. 0.5% agarose gel dissolved in fertilizer solution was added to each well of the 12 well pollinator plates until it solidified. Then the six pieces of stigma with a small part of style was added on the surface of the agarose gel. Pollen was added on top of the stigma and ovules were placed a few inches away from each stigma in each well.

In the bottom well, two stigmas without the style were cut into six pieces and placed on the surface of the agarose gel with pollen on top of stigma and ovules below. A fertilizer solution was added to hydrate the agarose gel and the pollinator plate was covered with a lid and incubated at Room Temperature (RT) for 24 hours. The wells were then observed for pollen tube growth under a light microscope with total magnification of up to 400X.

This was done to test whether the presence of ovules encourages more pollen tube growth.

Ovules placed a few inches away from the stigma

Tip of stigma with

pollen on top if it

Stigma with part of the style with overlying pollen

Figure 11 Stigma, pollen and eggs in wells

In the second variation, the space between the stigmas in each well was increased in case the pollen tubes require more room to grow. So, each well had only one stigma cut into three pieces. In the upper two wells in Figure 12, stigma tips with a part of the style were placed on top of the agarose gel with pollen overlaid on top of it while the isolated ovules were placed a few inches

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

away. In the lower two wells in Figure 12, were designed as the controls for the experiment. Each well had stigma tips without pollen and eggs placed a few inches away from the stigma. Ovules and pollen were also added together in a petri dish in agarose gel to observe pollen tube growth.

Stigma with part of style overlaid with pollen and ovule a few inches away

Control for the experiment without pollen

Figure 12 Stigma, pollen and ovules in wells with more room

Method 3: Pollen with whole pistil

In this method, the whole pistil with its stigma, style and ovaries were included instead of being cut up into pieces. Three entire pistils were placed on the surface of a 0.5% solidified agarose gel with fertilizer solution in a petri dish while pollen was added on top of the stigma. The petri dishes were covered with the lid at RT and then observed for pollen tube growth overnight.

Ovary

Style of the pistil

Stigma with pollen

Figure 13 Whole pistils with pollen in a petri dish with fertilizer solution and agarose gel

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

Method 4: Pollen and Ovules

This method involves deposition of pollen only on agarose gel with fertilizer solution from Wells 1-6 in the first row of a 96 well plate. The adjacent wells 7-12 were deposited with ovules on the surface of the agarose gel.

The second row of wells from 1-7 had both pollen and ovary on the 0.5% agarose gel supplemented with fertilizer solution. Wells 7 through 12 in the second row had only the agarose growth medium with nothing added and serves as the control. This experiment has been repeated for four times.

Figure 14 Pollen and ovules on the agar dissolved in fertilizer solution

Method 5: Johnson-Brousseau and McCormick Method

Sheila A. Johnson-Brousseau and Sheila McCormick describe a method for growing Arabidopsis pollen tubes [7]. They successfully grew Arabidopsis pollen using the method shown in Figure 14.

The same procedure was followed for Lilium lancifolium pollen with agarose gel pads made with 10% sucrose added to it. 1% Agarose was melted to which 130mg of sucrose was added and a few drops of this mixture was placed on a glass slide to form agarose pad. The agarose pad was cooled and 1-2 drops of pollen solution was added to each of the gel pad using a pipette tip. These glass slides were transferred to a pipette tip box that is humidified with water at the bottom.

13 Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

Kim wipes moistened with fertilizer solution were placed below the slides and the box was covered and incubated at RT for 2-3 hours and observed for pollen tube growth.

Figure 14 Johnson-Brousseau and McCormick Method to grow Arabidopsis Thaliana pollen tubes [9]

Nucleopore membrane overlaid with agarose gel and pollen

14 Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

Agarose gel pad on a glass slide with pollen on top of it pollen on top

Figure 15 Polycarbonate nucleopore membrane filter pads (top) and agarose gel pads (bottom) used with Lilium lancifolium pollen

Method 6: Yongxian Lu Method

The Yongxian Lu method [8].was a procedure used to track the pollen tube growth in- vivo in the female tissues after pollination. This method can be used to follow pollen germination, tube growth and guidance, and reception. The procedure outlines the stock solutions, and working solutions need in order to visualize the pollen tubes using a microscope with a UV light (in our case we used a handheld UV light). The 0.05% aniline blue (in 50 mM phosphate buffer, pH 7.5) can be used to view pollen tubes on the fluorescent microscope using a DAPI filter. To see the tubes on the interior of the pistil you will need to squash the tissue firmly with the cover slip. With this stain, the pollen grains (but not pollen tubes) also can be visualized with the rhodamine filter. The procedure was as adapted for visualizing the pollen tube growth in Lily as follows:

1. Submerge pistil tissue in 5-ml acetic acid and fix it for 1.5 h or more in a glass test tube (10-mm x 110 mm).

2. Soften tissue by submerging it in 1 M NaOH overnight.

3. Wash 3 times with KPO4 buffer. (Tissue is fragile at this stage)

4. Transfer to a slide, add mounting media (10ml Glycerol and 10ml 50mM KPO4) and observe under UV. Squash if necessary.

15 Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth Style Stigma

Figure 16 Stigma and style, with Aniline Blue staining, under a microscope to get background control and stain any other structures.

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RESULTS AND DISCUSSION

Method 1: Pollen and Stigma

No pollen tube growth was observed after the first two trials. In the third trial, a large amount of growth was seen. However, very high density of pollen tubes is not suitable since we cannot pick individual tubes for fertilization.

Figure 17 Dense pollen tube growth, as seen through a microscope (individual pollen tubes are not identifiable)

The experiment was repeated once more with lower pollen count but no pollen tube growth was observed. We had used lots of pollen on top of the stigma and instead a small quantity of pollen would have helped to visualize pollen tubes on top of the stigma.

Methods 2-6

In our hands, none of the methods 2 through 6 worked though we followed the literature using model like Arabidopsis thaliana for the growth of pollen tubes. There are countless literature sources demonstrating the growth of pollen tubes in-vitro as well as in-vivo but we were not able

17 Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

to duplicate their results. Failure to get the pollen tubes to grow outside of the plant in our experiments was probably due to many factors like the following:

1. Not getting the correct nutrient conditions for proper cellular growth

2. Not having the correct conditions that stimulate pollen tube growth by not being able to simulate the proper conditions (viscosity of plant polymers, nutrients, hydration conditions, etc.) inside the style of the plant

3. Not having a good method to determine the viability of pollen

4. Not having developed the proper techniques to selectively dye the pollen tube versus the surrounding plant materials

We tried a blue aniline staining method (Yongxian Lu) and congo red staining method (Edlund, A. and D. Preuss lab) [9] for the visualization of pollen tube growth (developed for visualization in Arabidopsis pistils) for Lilium lancifolium pistils. These protocols called for the visualization of the pollen tubes in the pistils using a confocal microscope with an excitation wavelength (543 nm) and observe after allowing the emitted light to pass through red filters (633 nm) for Congo red, or a fluorescent microscope using a DAPI filter to visualize the pollen tubes in aniline blue dye. In our set up we used a light microscope with a handheld UV lamp to visualize the dyes. It was effective for visualizing the aniline blue dye as seen in Figure 16.

In order to visualize the growth of pollen tubes in pistils and the visualization of pollen we will need to use either a fluorescent microscope with specialized filters or the confocal microscope with an excitation wavelength of 543 nm and a band-pass filter of 633 nm.

We will also need to optimize the aniline blue staining protocols (Yongxian Lu Method) or the Congo red staining protocols (Edlund, A. and D. Preuss lab) in order to optimally manipulate the pollinated style. Granted most of these protocols were optimized for the Arabidopsis T. and we were adapting them to visualize the pollen tube growth in the pistils of lilies, we still anticipate that these protocols are transferable to the larger plants.

The Arabidopsis, although widely researched because of its short reproductive and growing cycles, has very small sexual parts in which to work with. By using the larger sexual

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

parts of the Lily (anthers, pollen grains, stigma and style) it is much easier to handle and visualize the individual components.

In the Lily, the objective would have been to buy a flowering Lily at a local grower and manually fertilize the stigma with pollen taken from the anthers at different time points. Then each pollinated stigma and style would be excised from the flower of the Lily and processed according to the staining protocols developed by Yongxian Lu or Edlund and Preuss. Using two different staining protocols to visualize the growth of the pollen tubes, and the length of each pollen tube grows in a specific amount of time was thought to be a very interesting experiment which could be done in classrooms. We also thought that simple light microscope using a handheld UV lamp might also be used to visualize the pollen tubes in the style or the pollen grains. Now we know more sophisticated microscope with a powerful UV or fluorescent light source and specialized filters must be used to visualize the plant reproductive parts.

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

CONCLUSION

Though our experimental attempts to duplicate the growth of pollen tubes in Lilium lancifolium did not work either in-vitro or in-vivo, we need more time to develop and optimize the procedures. Also, we need a better UV and fluorescent microscope with more wavelength filters to visualize the dyes at different excitation and emission spectra. We plan to repeat the optimized method of staining by Yongxian Lu or Edlund and Preuss in the future for observing the pollen tube growth.

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

REFERENCES

[1] A K Yetisen, L Jiang, J R Cooper, Y Qin, R Palanivelu and Y Zohar “A microsystem-based assay for studying pollen tube guidance in plant reproduction” J. Micromech. Microeng. 21 054018 [2] A Krichevsky, S V. Kozlovsky, G-W Tian, M-H Chen, A Zaltsman, V Citovsky “How pollen tubes grow” Dev Biol. 2007 Mar 15;303(2):405-20 [3] T. Higashiyama, S. Yabe, N. Sasaki, Y. Nishimura, S. Miyagishima, H. Kuroiwa, and T. Kuroiwa, "Pollen tube attraction by the synergid cell", Science, 2000, 293, pp. 1480. [4] Mitsuhiro Horade, Yoko Mizuta, Noritada Kaji, Tetsuya Higashiyama, and Hideyuki Arata, "Plant-on-a-Chip Microfluidic-System for Quantitative Analysis of Pollen Tube Guidance by Signaling Molecule: Towards Cell-to-Cell Communication Study", microTAS 2012, Oct., Nov. Okinawa, Japan, pp. 1027-1029 [5] T. Higashiyama, S. Yabe, N. Sasaki, Y. Nishimura, S. Miyagishima, H. Kuroiwa, and T. Kuroiwa, "Pollen tube attraction by the synergid cell",Science,2000, 293,pp. 1480. [6] Alice Y. Cheung and Hen-ming Wu,”Pollen Tube Guidance--Right on Target” Science 24 August 2001: 1441-1442 [7] Sheila A. Johnson-Brousseau and Sheila McCormick, “A compendium of methods useful for characterizing Arabidopsis pollen mutants” Plant Journal 39: 761-775. [8] Yongxian Lu, “Arabidopsis Pollen Tube Aniline Blue Staining” http://www.bio- protocol.org/e88 (http://bio-protocol.org/wenzhange.aspx?kid=88)

[9] Emily Hartman, Clara Levy, David M Kern, Mark A Johnson and Amit Basu, “A rapid, inexpensive, and semi-quantitative method for determining pollen tube extension using fluorescence” Plant Methods 2014, 10:3

[10] http://kids.britannica.com/comptons/art-53831/Reproduction-in-flowering-plants-begins- with-pollination-the-transfer-of

[11] http://freewallsource.com/tiger-lily-flower-1343.html

[12]http://markbradley.pbworks.com/w/page/20366572/Angiosperms%20and%20Gymnosperms %20Assessment%20Task

Harika Sokkam, Aug 06, 2014 Evaluation of Pollen Tube Growth

[13] Minh Huynh, Pollen Growth Cell Fertilization Kit (Sea Coast Science)

Evaluation of Pollen Tube Growth using Lilium Lancifolium (Tiger Lily) Flowers

Harika Sokkam Sea Coast Science, Inc Introduction Sea Coast Science • Sea Coast Science is a company focusing on the chemical sensor and chemical detection market. • The company is also developing a new product called Pollen Growth Cell (PGC) kit, designed to study pollen tube growth in a lab/classroom environment.

Flowering Plant fertilization

About Tiger Lily

• Tiger Lily is a plant which belongs to Liliaceae family. • Tiger Lily’s stamen, style and stigma are much larger compared to Arabidopsis thaliana, which has been used extensively in related research. • We chose Tiger Lily due to it’s size which makes it easy to study the pollen tube in a class room setting

Lily

Tiger lily Arabidopsis thaliana (around 120 mm) (around 3mm) Fertilization using Pollen Growth Kit

• The project involves evaluating the growth of Tiger Lily pollen tubes in controlled lab environment using pollen growth kits. • Project aims to study and compare various methods for pollen tube growth for Tiger Lily. • We evaluate the usefulness of various methods to find the best one Collection of Pollen

• The first step in each method is the collection and storage of pollen. It’s important to use healthy pollen for the procedures to succeed. • Pollen was collected from anthers of the Tiger Lily flower. • Dried Pollen was transferred into a 5ml glass container with fertilizer solution and dilute laundry detergent solution.

Pollen being 5ml containers with collected from pollen in solution Anthers Separating Stigma, Style and Ovaries

Tiger Lily’s Stigma, Style and Ovaries after being separated from the rest of the flower.

. Method 1: Pollen and Stigma

• Each tip of the Stigma was cut into 3 pieces and pollen was delivered on top of the stigma

Tip of the Stigma Stigma being cut A third of the sigma after addition of pollen Method 1: Pollen and Stigma • Stigma and pollen were then transferred to wells with fertilizer solution • 2, 4, 6 trinitrobenzene sulfonic acid dye was used to help view the pollen tubes. The dye stains specific polymer elements in the pollen tubes.

Stigma with pollen in wells with fertilizer solution Method 1: Pollen and Stigma • No pollen tube growth was seen in the first two trials • Large amount of pollen tubes seen after the third try • The very high density of pollen tubes is not suitable. We cannot pick individual pollen tubes to use for fertilization • The experiment was repeated three more times with lower pollen density but no pollen tube growth was observed.

Dense pollen tubes under microscope Method 2: Pollen, Stigma and Ovules • This method included ovules in the growth medium to check whether the presence of ovules encourages more pollen tube growth • Extracted ovules from the ovaries using a Scalpel

Ovules being extracted onto a micro slide under microscope. Method 2A: Pollen, Stigma and Ovules • In the top well two stigmas were cut into six pieces. Pollen was added on top of the stigma and ovules were placed a few inches away from the each stigma • In the bottom well two pieces of stigma along with small part of the style was cut into six pieces. Pollen was added on top of the stigma and ovules were placed a few inches away from the stigma. • No pollen tube growth observed

Stigma, pollen and eggs in wells with fertilizer solution and agarose gel Method 2B: Pollen, Stigma and Ovules • Repeated the experiment with increased space between the stigmas in case pollen tubes require more room to grow • No pollen tube growth observed

Only 3 stigmas along with pollen and eggs in each well Method 3: Pollen with Whole Pistil • This experiment included the whole pistil with stigma, style and ovaries • Three pistils were placed in a petri dish and pollen was added on top of the stigma • No pollen tube growth observed

Pistils and Pollen in a Petri dish Method 4: Johnson-Brousseau and McCormick Method • Sheila A. Johnson-Brousseau and Sheila McCormick describe a method for growing Arabidopsis pollen tubes • They successfully grew Arabidopsis pollen tubes using 2 methods

Johnson-Brousseau and McCormick Method Method 4: Johnson-Brousseau and McCormick Method

• Tested Tiger Lily pollen with both agarose gel pads and membrane filters • No pollen tube growth observed

Polycarbonate nucleopore Agarose gel pads with pollen on Membrane filter pads top of it Method 5: Yongxian Lu Method • Tested the pollen tube growth method as described by Yongxian Lu. • Used various chemicals and reagents along with staining dye to track pollen tube growth and performed the procedure • Stigma and style were also stained separately to be used as a control to identify staining due to pollen tube growth

Stigma and style, with Aniline Blue staining, Style with aniline blue stain. under a microscope Results and Discussion

• No repeatable pollen growth was obtained using any of the methods • Failure to get the pollen tubes to grow outside of the plant in our experiments was probably due to many factors like the following • Not getting the correct nutrient conditions for proper cellular growth • Not being able to simulate the conditions inside the style like viscosity of plant polymers, nutrients, hydration conditions, etc • Not having a good method to determine the viability of pollen • Not having developed the proper techniques to selectively dye the pollen tube versus the surrounding plant materials

Conclusion

• Many successful prior studies have been conducted using the methods mentioned • This gives us confidence that given enough time, we will be able to develop and optimize the procedures for Tiger Lily. • We plan to repeat the optimized method of staining by Yongxian Lu or Edlund and Preuss in the future for observing the pollen tube growth.

References