The Adventure of Research

The sterilization protocol and culture for the sampling of T. testudinum produced highly contaminated cultures. Standard sterilization procedures commonly used for terrestrial plant material were insufficient for obtaining axenic Thalassia testudinum. It is important to remark that for each different culture medium there was a control tube that was subjected to the same conditions of experimentation and none of this control tubes showed contamination. This fact indicates that the contamination observed in the culture tubes must come from within the tissue. Surface and endophytic bacteria, as well as fungi, are prevalent on seagrasses (Ailstock, 2006). This diversity of associated microorganisms, coupled with the characteristic lack of a well-developed cuticle in submerged plants (Koch, 2001), makes effective, non-deleterious surface sterilization difficult (Ailstock, 2006). The major obstacle for developing tissue cultures of submerged plants is the difficulty in obtaining axenic cultures. The multi-step sterilization protocol used here produced no axenic plants.

This can occur because both the fungicide and antibiotics are not able to remove all the present contamination, and only go through the outer layer of the rhizome. In previous trials the fungicide employed was Captan®, but for this trial Terraclor® (PCNB) was used; it is possible that the correct amount of fungicide was not used or it was not managed appropriately.

Also, tissue growth was observed at first but after several weeks was not observed any kind of tissue development. There was no growth of stems, leaves or roots, and then the tissue became dark brown and began to die. (Koch, 2001). The malfunctioning of the incubator could be one of the reasons for these results. It is important to provide the optimal growth conditions or parameters for the explants; but the incubator temperature controller does not work properly causing higher temperatures than desired.

DNA extraction

The DNA extraction made for the apical meristerms of T. testudinum yielded low DNA concentration. It is necessary to optimize these protocols to determine the endophytic fungal diversity present in T. testudinum rhizomes by culture independent techniques.

Recomendations

1. Perform various trials to determine the correct amount of fungicide needed to produce axenic explants.
2. Use only apical meristerms for T. testudinum in vitro cultures
3. Optimize the DNA extraction protocols
4. Work with the technical problems of the incubator
5. Further research is needed to identify the fungi species and analyze if a cryptic microbial community persist within micropropagated T. testudinum.

Marrero – Berríos, Ileana¹ & Maldonado – Ramírez, Sandra L., PhD²

Abstract

Thalassia testudinum is the most common and abundant seagrass in the Caribbean; but since the 1800s, approximately 80 percent of the seagrass coverage has been lost mainly due to human activities. An effective method to propagate submerged plants in a short time is needed such as micropropagation, till these days no one has micropropagated T. testudinum under axenic conditions. Micropropagation of plant species is the name for the different sterile techniques and strategies that make possible the development of new plants from cells, tissues and organs. The major obstacle for developing tissue cultures of submerged marine plants is the difficulty in obtaining axenic cultures. Tissue samplings of T. testudinum were obtained from Buyé Beach at Cabo Rojo, Puerto Rico.  Several sterilization protocols and various Murashige and Skoog’s medium concentrations were used in terminal rhizome segments, bearing apical meristems and three to five nodes in an attempt to obtain axenic T. testudinum explants. Standard sterilization procedures commonly used for terrestrial plant material were insufficient for obtaining sterile axenic T. testudinum explants.  Surface and endophytic bacteria, as well as fungi, were prevalent on the seagrass tissue sampled. An additional aim of this project is to determine the fungal endophytic diversity of T. testudinum rhizomes by culture dependent and independent techniques.   Further research is needed to develop a reliable sterilization protocol, to determine the precise nutrient requirements to develop viable, uncontaminated explants, and to determine the fungal endophytic diversity on T. testudinum rhizomes. This research was sponsored by BioMINDS program.

Results

Standard sterilization procedures commonly used for terrestrial plant material were insufficient for obtaining axenic Thalassia testudinum. Surface and endophytic bacteria, as well as fungi, are prevalent on seagrasses . This diversity of , makes effective, non-deleterious surface sterilization difficult (Ailstock, 2006).The major obstacle for developing tissue cultures of submerged plants is the difficulty in obtaining axenic cultures. The multi-step sterilization protocol yielded 13 axenic Thalassia testudinum from the original 32 processed rhizomes (41%). Contamination, when present, was mostly due to fungi, which tended to grow on the surface of the medium. Slow growing endophytic bacteria, presumably residing in the relatively large explants, may also have been present because, in several cultures, the medium did not become cloudy until after several weeks of culture.

Isolation and identification of prevalent fungi in T. testudinum explants

Fungi present in the explants samples were isolated and studied; continuously finding the genera Cladosporium sp. and Penicillium sp. Penicillium spp. is the most frequently associated genera with infected explants

Light microscopy photographs of Penicililum spp1 (A), Penicillium ssp2 (B), Penicillium spp3 (C), and Cladosporium sp (D).

Isolated Penicillium sp

Isolated Cladosporium sp.

Also, tissue growth was observed at first but after several weeks was not observed any kind of tissue development. There was no growth of stems, leaves or roots, and then the tissue became dark brown and began to die.

For these semester Theodor is planning to continue using the RT-PCR technique to confirm the expression or oppression of the XFBXO30 protein in the different embryo stages.  In addition, he wants to learn and practice the 5’RACE technique, so he and his laboratory team can obtain the 5’ end of the XFBXO30 gen sequence.  Also he will learn and practice in vitro fertilization and RNA microinjection into eggs and embryos! I think that is really awesome!

Xenopus laevis

The second BIOblog I visited was Germán Vélez’ s Blog. Germán’s research project is about the Human Papiloma Virus (HPV). His lab’s team wants to know the frequency of HPV16 E6 and E7, exons that are present in cancer patients in Puerto Rico. This information is very important for them because it will drive his team and other scientists to new research and questions.  Once his lab team decipher the frequency, they want to know the exact type of HPV16, this is the exact mutation in the DNA.

During this semester Germán hopes to finish the detection of HPV16 in Head and Neck Cancer, and he will begin the detection of HPV16 variants. Some of his goals are: complete detection of HPV16 variants, develop new protocols and a new data base with easy access, and improve his knowledge in molecular biology.

Human Papiloma Virus

      Besides continuing with the various protocols of micropropagation, I am interested in researching the different encrypted microbiological communities that inhabit in the T. testudinum processed tissue. To perform this type of study is necessary to conduct a fungal DNA extraction. To do this there is no standard process, we must try different combinations to find the correct amount of tissue DNA extract as much as possible. Also, I am not using a commercial extraction kit, all components to make the extraction are prepared in the laboratory by varying the concentrations of these until you find the correct protocol!

T. testudinum apical meristerm growing

My lab's aquarium... yes that's me!

The first BIOblog I visited was Doris Abreu’s blog; her research project is about a biosensor. This biosensor is an aptamer that binds specifically to a tumor marker protein named VEGF which stands for Vascular Endothelial Growth Factor. The studied aptamer is the VEa4. When this aptamer binds to the protein, it can work as a biosensor and it can be used for the diagnosis of different diseases such as cancer. The importance of this work towards the scientific community is that if the secondary structure of the aptamer VEa4 and the protein VEGF₁₆₅ are determined and analyzed, the bonds that are formed between them can be studied and used to detect if a patient has a vascular disease or not. Then a treatment can be synthesized on time so the probabilities of the patient’s survival will be much bigger.

The second BIOblog I visited was José Pagán’s blog. He is a new member in the BIOMinds Program and also is a Chemistry freshman student on UPR Cayey! That is what I call an early start! For this semester his work was to search, find, and study scientific literature to acquaint with important subjects of the research. E.g. Analytical techniques like: Gas Chromatography Mass Spectrometer “GC/MS”, volatile compounds, functional groups, and, calibration standards. He was in charge of the optimization of the GC/MS system, specifically to accustom with the hardware, and software programming. Also as part of his work, he had to calibrate the equipment using calibration standards.

The third BIOBlog I visited was Nerymar Ortiz’blog; her research project is aboun Sulfhemoglobin. This is a dangerous compound because when hemoglobin contains sulphurous it does not allow the protein to transports oxygen. Weather this occur, the organism die by a deficient amount of oxygen. Between her tasks were: determine factors that control the formation of the sulfhemoglobin complex using UV-Vis spectroscopy and HbI mutants; determine the optimal conditions for a best formation and stability of the sulheme complex; and know the mechanism of reaction in sulfhemoglobin derivates formation to help treat the clinical condition known as sulfhemoglobinemia.

Last post of the semester:

This semester I learned so many things about how a research laboratory works. A new graduate student began her thesis project on my research topic and I was training her on different techniques and aspects in the lab such as in vitro cultures, sterilization protocols, media preparation, isolation and inoculation of mycelial fungi, etc. Also I had the opportunity, as a BIOMinds student, to participate in the Sigma Xi Poster Day; this was very enriching for me because I learned how to prepare and present a poster.

This semester was not so productive; we were stock in our research because some materials arrived in March. But we carried a third trial on in vitro cultures for T. testudinum on liquid media and the results were very interesting.  Aerophilc fungi when present did not damaged or infected the rhizomes tissue, on the contrary, the tissue was healthy.

One of the techniques I learned during my classes and laboratories is the extraction and amplification of DNA. I will use this technique in my project to make a more specific study of the types of fungi that Thalassia testudinum have encrypted inside the leaves. This experiment will be conducted with normal leaves and leaves after processing them with the rigorous sterilization protocol that is used to cultivate them in vitro. After extracting the DNA it will be sequenced to ensure that we have in the “endophytic fungi library” all the cultivable species that are present inside T. testudinum leaves.

Using a scale of 1-5; I would say that my project has only a 2 because wrong materials came to the laboratory and we had to wait several weeks for the correct. But we already have the aquarium system with T. testudinum growing , one of our goals from the beginning.

Aspergillus sp.

Hello!!!! Welcome to my blog!

For this semester our laboratory team has the same objectives of last semester, but we are trying new ways to reach our goal. Our most important task is to obtain axenic cultures of Thalassia testudinum to study mutual relationships between the endophytes and the plant.

At this time, we are collecting samples; on Buye Beach in Cabo Rojo, Puerto Rico, looking for flowers, seeds and plants of T. testudinum. Last semester we were doing in vitro cultures of tissue from the rhizomes, but for this semester will use tissue from flowers, leaves, rhizomes and seeds. We will do these various tests to see which tissue is best developed and which has less population of microorganisms.  Also, we will be changing the pH and concentration of growth factors in culture media. All these arrangements are being made to optimize the growth conditions of axenic T. testudinum.

During this semester I will develop several skills like culturing tissue at a cellular level, working with new protocols for sterilization, preparation of media for different types of tissue, and work in microscopy using scanning and transmission electron microscopes.

Thalassia testudinum figure

The first bio-blog I visited was Josue Negron’s blog. He is an UPRH biology student and is working with new amphibian species. Right now his team is documenting the basic reproductive parameters of a recently discovered Coqui, Eleutherodactylus juanariveroi. Also they are orienting the community about these new species, and I believe this is very important so they have the knowledge that is a need to preserve this species.

Last post, November 2009!

One of the most important lessons that I have learned this semester is to be patient and perseverant. In vitro plant tissue culture is a process that has to be carried in extremely sterile conditions and the person working with the tissue (me) has to be very careful to avoid tissue damage. Also I have to wait long time in order to obtain positive results (tissue growth and no contamination).

At this time my second trial on in vitro cultures of Thalassia testudinum is growing very well! I am making new nutritive medium with different concentrations of auxines and cytokinins (auxines are hormones that affect plant growth, they can be characterized by their ability to induce cell elongation in stems, and meanwhile cytokinins are compounds which promote cell division), to transplant the tissues with the best development range and determine the best growth conditions for the T. testudinum tissues.

My next semester is full of hard work from beginning to end! Once we have all the tissue growing in the required conditions, a system of little aquariums would be installed and part of the plants will be put in there, in order to induce propagation of the sterile clones, and the other part will be use in different tests with endophytic fungi to determine their function in the plant.