Genetic Arbidopsis Lab

Abstract

            The purpose of this lab experiment is to observe the mechanism of the leaf morphogenesis through the characterization and isolation of two mutants of Arabidopsis thaliana. We observed the various characteristics of the wild type Arabidopsis and the mutants. We started with one wild type phenotype (Col), which is round, short and regular with hairy trichome. We also observed and recorded the phenotypes of the two mutants; (gl1) and (CA1). We first crossed the (ColXCA1) and then (ColXgl1) to obtain the F1 seedlings. However, the cross was not successful and we had to wait to get the F1 seedlings from the other students.

In the F1 phenotype for (ColXCA1), we found the serrated edges with hairy trichome were dominant, and for the cross (ColXgl1) oval elongated and hairy trichome that belonged to Col, were dominant. After getting the F1 seedling, we had to look for the F2 phenotypes, and calculate the chi square to analyze our result. Our results were acceptable, since we had a ratio of 3:1. In conclusion, we felt that this was a very good experiment to develop necessary skills in genetic analysis, to understand the Mendel’s basic genetics law of equal segregation and to observe the morphogenesis of different plants, including the wild type (Col) and the mutants (CA1), and (gl1).

Introduction

            Arabidopsis thaliana is a weed plant that belongs to the brassicaceae (cabbage) family of plants and it has become the model plant of genetics since 1980s. Although closely related to such economically important crop plants as turnip, cabbage, broccoli, and canola, Arabidopsis is not an economically important plant. Despite this, it has been the focus of intense genetic, biochemical and physiological study for over 40 years because of several traits that make it very desirable for laboratory study. As a photosynthetic organism, Arabidopsis requires only light, air, water and a few minerals to complete its life cycle. It has a fast life cycle, produces numerous self progeny, has very limited space requirements, and can be easily grown in a greenhouse or indoor growth chamber. Because of its short genome size, the genome of the Arabidopsis is sequenced completely.

Arabidopsis thaliana, a small flowering annual dicotyledonous plant, was discovered by Johannes Thal (hence, thaliana) in the Harz Mountains in the sixteenth century. “However, it is only since 1943 that Freidrich Laibach reported for the first time the potential of this plant as a model organism for genetic studies (W. David et al., 1998.).” The Arabidopsis is a distinct plant that takes a while to germinate. Arabidopsis plant is naturally distributed all throughout Asia, Europe, and North America. Over the years, more than 750 natural accessions of Arabidopsis thaliana have been collected from different parts of the world. There are many reasons why geneticists choose it as a model plant over others plant organism.

Arabidopsis has a very short life cycle of about 6 to 8 weeks. It is capable of self-fertilizing and is easily transformed. Each plant has about 5000 seeds, which serve as genetic stock. Arabidopsis has five pairs of chromosome. It has one of the shortest genome (125Mb) among all other plant. In this experiment, we are basically observing the leaf morphogenesis of the wild type Arabidopsis and the two mutants. Leaves are very important organs of a plant. Leaves are the main component for photosynthesis, and control the air/water exchange. Plants have different shapes of leaves in order to sustain in constantly changing environment, such as light, CO2, water. “In many species, plant pairs, trichomes are believed to provide physical protection from attack by predators” (Daniel et al., 1998).

Trichomes are specialized epidermal cells that are regularly distributed on the leaves of Arabidopsis plants. In Arabidopsis leaf trichomes are unicellular and stellate. “More than twenty genes are required for the correct initiation, spacing, and morphogenesis of trichomes in Arabidopsis” (Daniel et al., 1998). Although, more than twenty genes are required for the correct initiation of the trichomes, mainly two key genes GLBROUSE1 (GL1) and TRANSPARENT TESTA GLBRA (TTG) are required for trichome initiation. “Genetic experiments showed that both GL1 and TTG activity are required for trichome initiation” (Daniel et al., 1998).

Although, the overall effect of GL1 overexpression in the leaf is initiation, GL1 overexpression also leads to limited trichome initiation on the cotyledon. There are some other kinds of genes that partially affect the initiation of the trichome initiation. “The GLABROUS2 (GL2) gene is required for subsequent phase of trichome morphogenesis such as cell expansion, branching, and maturation of the trichome cell wall” (Daniel et al., 1998). “Mutations in the GL1 alleles affect only trichome development. Mutations in the TTG locus are pleitropic responsible for a diversity of processes. Mutant plants lack trichomes, seed coat mucilage, and anthocyanin pigments. Also ttg mutants show an increase in the production of root hair” (Daniel et al., 1998)

            So far, geneticists have isolated and characterized more than 70 trichome mutants that represent 21 different kinds of genes. By analyzing their cellular phenotypes, it has been possible to define specific steps in trichome development (David et al., 1998).

Materials and methods

            In our experiment, we used three Arabidopsis plants. Among those plants one (Col) was wild type, and other twos gl1, and CA were mutants. We observed and then recorded the phenotypes of these three plants genotype. Firstly, we crossed Col (female) with CA1 (male) in one segment, and then we crossed Col (female) with gl1 (male) in another segment. For these crosses to be carried out we emasculated the flower buds to prevent self pollination. To perform this we used a pair of extra fine-pointed forceps, and scissors for cutting. We also used a stereo microscope for making the right cuts around the stigma of the flower.

We had to be very careful when we opened the pistil, because damaging pistils would have resulted in the failure of growth and so the failure of fertilization. In contrast to the pistil, the removing of the sepals and petals were relatively easy. After that, we had taken a fresh, fully open flower from the male parent and rubbed on the stigma of the female plant. We did this procedure under the microscope we had to make sure that the crosses were performed according to the instruction. Before we made any new cross, we sterilized the forceps with 70% ethanol. At the end, we labeled the plant crosses accordingly- Col x CA1 and Col x gl1.

F1 characteristics we observed

            My partner and I were very careful to perform the crosses. Unfortunately, despite our effort, our crosses were not successful, hence aborted. So we asked the students from the next group, who had successful crossed, to give us the F1 seedlings. We used these F1 seedlings throughout our experiment. We then sowed the F1 seedlings for obtaining the F2 seedlings. After getting the F2 seeds, we germinated these seeds to obtain the final scoring of the F2 phenotypes. In the below table I summarize the result of our F1 phenotypes results.

                                               Arabidopsis flower plant

     Results of F1 generation

Crosses	Number of flower buds crossed	Number of siliques obtained	Number of F1 seedlings obtained	F1 seedling phenotype
ColxCA1	–	–	10	Serrated edges with hairy trichome
Colxgl1	–	–	14	Oval elongated with hairy trichome

            Since our crosses were not successful, we could not collect all the necessary data, like number of flower buds, number of siliques etc. for our lab experiment. But we collected the number of F1 seedlings that shown in the table. In table 10, plants were present with serrated edges and hairy trichome, showing the phenotype of CA1. On the other hand, 14 seedlings with oval elongated and hairy trichome, showing the phenotype of Col.

Results of F2 generation

Crosses	Rounded hairy	Rounded hairless	Narrow hairy	Narrow hairless
ColxCA1	20	–	53	–
Colxgl1	44	17	–	–

The above table shows the results of the F2 generation Arabidopsis plants.

Arabidopsis wild type hairy        Arabidopsis gl1 mutant, hairless   Arabidopsis ttg mutant

Chi-Square analysis/ Results

                                      Chi-Square Analysis for cross (ColXCA1)

ColxCA1	O	E	O-E	(O-E)^2	(O-E)^2/E
Round & Hairy	20	18.25	1.75	3.0625	0.167808
Narrow & Hairy	53	54.75	-1.75	3.0625	0.055936
				Total X ^2 Value	0.223744

            Degree of freedom: one

            Based on our Chi-Square=0.2237 (In the above table) we can accept the null hypothesis of dominance of narrow hairy over the round hairy. Therefore, the round hairy in this case is recessive. P-value was found to be in the 25%-50% region and this easily supported the law of segregation in our experiment.

Chi-Square Analysis of cross (ColXgl1)

Colxgl1	O	E	O-E	(O-E)^2	(O-E)^2/E
Round & Hairy	44	45.75	-1.75	3.0625	0.06694
Round & Hairless	17	15.25	1.75	3.0625	0.20082
				Total X ^2 Value	0.26776

            Degree of freedom: One

            In this case, based on our chi-square analysis in the above cross (ColXgl1), we accept our hypothesis of the dominancy of round and hairy over the round and hairless. The Chi-Square=0.26776 and the p-value in the region of 25%-50% tests support our hypothesis for law of equal segregation.

Discussion

            Arabidopsis thaliana is a wonderful plant that has been the subject of intensive research over the years, due to its short genome, very short life cycle and many others advantages. Due to its easy visibility, this small angiosperm can serve as a wonderful model for addressing fundamental questions of biological structure and function common to all eukaryotes. After observing the results, we figured that there is no linkage; rather equal segregation of genes is available for both crosses we made in this experiment.

In the cross between (ColXCA1), there are two genes that are functioning and, narrow and hairy is dominant over rounded hairy. In the cross (ColXgl1), round and hairy trait shows dominance over the round and hairless. The only difficulty we had in this experiment to not get a successful cross for F1 generation, but we overcame this problem and overall our lab experiment was successful.

Conclusion

            In conclusion, I can say that the main purpose of the lab experiment was to observe the mechanism of leaf morphogenesis in Arabidopsis thaliana plant and observe the characteristics of various Arabidopsis plants like wild type (Col), and two other mutants (gl1) and (CA1). Through the appropriate techniques provided by our instructor, we were able to work out whether the mutants are dominant or recessive and observe the segregation of genes.

References

Szymanski, B. Daniel., & Marks, M. David. (1998). Glabrous1 Overexpression and triptychon Alter the Cell Cycle and Trichome Cell Fate in Arbidopsis. American Society of plant Physiologists. Vol.10. pp. 2047-2062.

Szymanski, B. Daniel., Jilk, A. Ross., Pollock, M. Susan., Marks, M. David., et al. (1998). Control of GL2 expression in Arabidopsis leaves and trichomes. Plant Molecular Genetics Institute, University of Minnesota, St. Paul. Vol.126. pp. 61-1171.

Meinke, W. David., Cherry, J. Michael., Dean, Caroline., Rounsley, D. Steven., Koornneef, Maarten. (23^rd October, 1998). Arabidopsis thaliana: A Model Plant for Genome Analysis. Vol. 282. pp. 678-681. Www. Sciencemag.org.