Evaluation of Pollen Tube Growth from Liliaceae Flower
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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 i 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 ii 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. iv 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. 2 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