Fascinating experiments with plants. by PubliUle

Fascinating experiments with plants

Fascinating experiments with plants José Luis Acebes Arranz María Luz Centeno Martín Antonio Encina García Penélope García-Angulo (Coordinators) Bridget Ryder (Translator)

Fascinating experiments with plants / José Luis Acebes Arranz... [et al.] (coordinators) ; Bridget Ryder (translator).– [León] : Universidad de León, [2021] 164 p. : il., fot., gráf. ; 30 cm En cub.: Ideas to immerse yourself in the fascinating plant world from an experimental point of view applied to teaching ISBN 978-84-18490-07-1 Plantas-Experimentos. I. Universidad de León. II. Acebes Arranz, José Luis. III. Ryder, Bridget 581.08 De acuerdo con el protocolo aprobado por el Consejo de Publicaciones de la Universidad de León, esta obra ha sido sometida al correspondiente informe por pares ciegos con resultado favorable. Reservados todos los derechos. Ni la totalidad ni parte de esta publicación pueden reproducirse, registrarse o transmitirse, por un sistema de recuperación de información, en ninguna forma ni por ningún medio, sea electrónico, mecánico, fotoquímico, magnético o electroóptico, por fotocopia, grabación o cualquier otro, sin permiso previo por escrito de los titulares del copyright.

ISBN: 978-84-18490-07-1 Depósito Legal: LE-25-2020 Diseño, maquetación y tratamiento digital de las imágenes: Juan Luis Hernansanz Rubio Imprime: Gráficas RIGEL Impreso en España / Printed in Spain Enero, 2021

We want to thank those who have contributed in one way or another to this publication finally seeing the light: The Faculty of Biological and Environmental Sciences, who embraced this teaching initiative and greatly assisted us in carrying it out. The Teacher Training School of the University of León, which year after year has financially supported the development of the experiments through their Teaching Innovation Projects grants. The undergraduate volunteers who participated in the activities of the Fascination of Plants Day and who year after year helped make improvements to the experiments collected in this book. We want to especially mention those who produced the videos included in the video channel Fascinating Experiments with Plants, which is an ideal complement to this publication: Verónica Alonso, Rosa Blanco, Ana Diez, Sofía Fernández, Alejandra Gallardo, Sara Martínez, Santiago Michavila, Víctor Moreno and Sarah Panera. To the primary and secondary school teachers and students who attended the workshops and gave us their feedback. To the Publications Service of the University of León, and in particular to Dr. José Manuel Trabado, for warmly accommodating this initiative. We also want to thank the two reviewers who evaluated the manuscript. Their reports have contributed significantly to its improvement. Finally, to all who have contributed ideas, initiatives, or have expressed their enthusiasm. Their "Wow!" or "How cool!" have been the definitive motivation to put these experiments down on paper.

PRESENTATION: THE FASCINATION OF EXPERIMENTING WITH PLANTS Pág. 11

1. FASCINATING MOLECULES

(Romina Martínez Rubio & María Luz Centeno Martín) Pág. 17 1.1. Obtaining visible DNA. Extraction of DNA. Pág. 22 1.2. Ethylene and fruit ripening. Ripe fruits produce ethylene. Pág.26

2. TRANSPIRATION: THE WATER LOSS CONTROL (Laura Pascual Vallejo & Penélope García-Angulo) Pág. 33

2.1. Pores that regulate hunger and thirst. Observation of stomata under a microscope. Pág. 37 2.2. How much do plants transpire? A measure of transpiration. Pág. 41 2.3. Designing an artificial plant. Transpiration and xylem transport. Pág. 45

3. PHOTOSYNTHESIS IN ACTION

(Alba Manga Robles & Antonio Encina García) Pág. 49 3.1. Blade discs with oxygen floats. Photosynthesis in action. Pág. 52 3.2. Experimenting with the colour of light. Does green light or red light have the same effect? Pág. 56 3.3. Timing photosynthesis. Is it possible to quantify the intensity of the process? Pág. 58

4. STARCH: A POLYVALENT COMPOUND

(Néstor Prieto Domínguez & María Luz Centeno Martín) Pág. 61 4.1. Is there starch in my mortadella? "The Lugol’s test". Pág. 66 4.2. What plant does this flour come from? Amyloplast observation. Pág. 70 4.3. Building a "ripeness meter". How ripe are my bananas? Pág. 75

5. THE FASCINATING COLOURS OF PLANTS

(María del Carmen Humanes Jurado, Laura Lindo Yugueros, Laura Pascual Vallejo & José Luis Acebes Arranz) Pág. 79 5.1. The purple cabbage indicator. A pH meter hidden in purple cabbage. Pág. 84 5.2. Vegetable art. Painting on papers coated with purple cabbage extract. Pág. 87 5.3. A brightly coloured tree. Painting on a purple cabbage leaf. Pág. 91

6. FASCINATING EXPERIMENTS WITH INVISIBLE INKS (Cristina del Amo Mateos, María Quevedo Araus & José Luis Acebes Arranz) Pág. 97 6.1. Writing invisible messages with potato. Starch revealed by iodine vapours. Pág. 102 6.2. Invisible writing with lemon juice developed with heat. Pág. 105 6.3. The purple cabbage spy. Revealed with purple cabbage extract. Pág. 108

6.4. Writing with white flowers. Developing with UV light. Pág. 111

7. DYNAMITE PLANTS. EXPLOSIVE VEGETABLES (Romina Martínez Rubio, Diana Mieres Roza & José Luis Acebes Arranz) Pág. 115 7.1. Exploding balloons with orange peels. Properties of essential oils. Pág. 119 7.2. Explosive vegetables. The power of catalase. Pág. 122 7.3. A lemon bomb. Lemon juice and bicarbonate: an explosive mix. Pág. 126

8. THE MOVEMENTS OF THE PLANTS. EXPERIENCING THE TROPISMS

(Iris Asensio García, Samuel Huerga Fernández, Carlos Frey Domínguez & Penélope García-Angulo) Pág. 129 8.1. Obstacle phototropism. Making turns through phototropism. Pág. 133 8.2. Moving to the sound of colour. Phototropism and the quality of light. Pág. 137 8.3. Plants in Z. Observing gravitropism. Pág. 142

9. THE MICROSCOPIC WORLD OF POLLEN

(Santiago Michavila Puente-Villegas & Laura García-Calvo) Pág. 147 9.1. Pollen grains: filigrees under a microscope. How to study pollen types. Pág. 152 9.2. Is my honey really made from a thousand flowers? The honey pollens. Pág. 161

Presentation THE FASCINATION OF EXPERIMENTING WITH PLANTS

PLANTS,

THOSE FASCINATING BEINGS

We are so used to living with plants, that we don't usually give them the attention they deserve. Imagine if plants and their products suddenly disappeared. Flop! This book would disappear in your hands! The paper it is printed on comes mostly from pulp made from wood and starch, which, of course, come from plants. The table and chair —or the armchair— where you are sitting would also vanish, and with it, the other furniture in the room, since the wood they are built with comes from trees. And what about our clothes? The cotton for our shirts and jeans comes from the seeds of cotton plants. Not to mention food, since it comes directly from plants, or from animals that feed on plants. Then there are also many of our medicines, perfumes, industrial products, biofuels... What if we considered parks and green areas? As one of the children in

our workshops said one day: "Let's see if I understood correctly. Without plants we cannot eat, dress, travel, or even breathe... Then without plants, we all die!" Our life depends in a very substantial way on plants. Let’s remember that, in absolute terms, every oxygen molecule we breathe has been produced in a chloroplast, and that the organic molecules we consume were initially generated also in chloroplasts.

Defining fascination Fascinating, according to the Oxford English Dictionary, means "extremely interesting" or “captivating”. Plants fascinate us, they are "extremely interesting" to the point of capturing our attention. They captivate because they are amazing.

Among these captivating powers of plants are fatal attractions, such as those of carnivorous plants that draw insects toward them with their colours and aromas and then... There are also other less dangerous attractions that result in a mutual benefit, such as those felt by pollinators for the flowers they visit. Plants also attract us humans with their colours, aromas, and shapes; their beauty; and their diversity. That is why we say plants are fascinating, and the more we know them, the more we admire them, and the more fascinated we become with them.

Experiencing fascination The capacity for admiration is one of the characteristics of the human species. The careful observation of our surroundings causes us astonishment. In children, this ability to marvel appears very soon. They are surprised by the almost infinite number of stars on a summer night, by the colour of a poppy, or by the slow walk of an insect. Later, in many people this fascination with the natural environment becomes deeply intellectual. It turns into a capacity for more acute observation, joined with emerging questions and the ability to develop procedures and resources to try to answer them. This is the beginning of a scientific vocation, which in some of these "captivated" by plants, will lead to higher studies.

valuable and useful. From "Look, Dad, the sky is full of stars!" to "See how it moves!" But communicating requires a certain technique. It requires experimenting, looking for the most appropriate method. Effective communication must be vital, contagious, fascinating.

“How cool!” One of the most rewarding moments in the educational process occurs when the teacher receives from a student the spontaneous exclamation of their fascination with being surprised by an experiment: "How cool!", "Wow!" It can also come when a student expresses at the end of a laboratory session: "What a fantastic afternoon we have spent!" These students have been bitten by the "bug of fascination". This sting is contagious, and is transmitted from one student to another. In it is the basis of another fascination, which we can call seduction by teaching. Teachers catch a glimpse of it when young people "waste time" to dedicate it to others and communicate to them what excites them. They are the protagonists. They do the staging. The little details. The practice their experiments before, to see where they can add a "twist,” or create a “Bang” that arouses a "Wow!" from the audience. Well, the book in front of you was born from and grew out of the experience of "How cool!"

Sharing the fascination At the same time, with the capacity for admiration, the need arises in human beings to tell others what fascinates them, everything that they discover and consider

The Fascination of Plants Day Instituted by the European Plant Science Organisation, May 18th is the International Day of the Fascination of

Plants, known by the acronym FoPD (see https://plantday18may.org). It is an initiative launched by the European Plant Science Organization (EPSO) aimed at raising awareness of the importance of plants for the present and future of our planet, and to highlight the fascination we feel for them. So far five official editions have been held, in 2012, 2013, 2015, 2017 and 2019. The next was scheduled for 2021, but it has been postponed to 18th May 2022.

of the activities are organised by small groups of volunteer students —some of whom are authors of the different chapters of this book— under the tutelage of a teacher. These experiments are the basis of this publication. With a few exceptions, all of them have the following characteristics: • They work. They have been derived from experience. They have been fine-tuned and repeated and repeated again. • They are active. Whoever does the experiment becomes an actor, taking an active part in the experiment. It is not only about observing curiosities of plants, but about working with your hands, your eyes, your head —in short, all the senses— to uncover the surprising facets of plants.

Figure 1. Group of scholars working on a workshop “Fascinating experiments with plants”, supervised by two monitors.

To cite some data, in the 2019 edition, 862 events were organized in 51 countries worldwide, promoted by very diverse institutions, including botanical gardens, universities, town halls, research centers, etc. Almost 10 percent of the activities (89 in total) were organized in Spain by 48 different institutions.

• They are fascinating. They are made to elicit amazement at and fascination with plants. They teach students about the unsuspected properties of plants and, sometimes, the amazing applications of those qualities in daily life. • They are simple. They require only fairly basic equipment. Only a few of them require a microscope or a laboratory reagent, but ones that schools usually already have.

Fascinating experiments with plants

• They are fast. They can be done in a short amount of time. Most require less than half an hour.

One of these events, which has been held since the first edition of the FoPD, is the Workshop Fascinating Experiments with Plants, sponsored by the Faculty of Biological and Environmental Sciences of the University of León (Figure 1). Each

• They are didactic. Each one ends with a brief reflection and to help students reach a conclusion about some aspect of the operation of the plants involved in the experiment.

• They are versatile. They have been written with secondary students in mind, but those same experiments, with small adaptations, are also useful for primary students (we have verified this on multiple occasions, see Figure 2). The experiments can also be very useful for university students. In the Learn More section at the end of each experiment, interesting data is provided to complete or to make the experiment performed more rigorous. Many of these experiments are also available on video. They can be found on YouTube, on the video channel: "Fascinating experiments with plants".

Fascinating experiences These activities are intended above all to provide ideas to immerse yourself in the fascinating world of plants from an experimental point of view. From a teaching perspective, depending on the context, they can be used to introduce or support the scientific method, to encourage the creativity of the students, to stress some theoretical or applied aspect of the plants, or to simply leave them astonished.

Figure 2. Primary students working with invisible inks obtained from plant extracts (See chapter 6).

In our teaching practice we have had the opportunity to verify that the greatest enemies of the educational process, for both students and teachers, are routine and reluctance, together with discouragement. The experiments that we propose here have contributed to a good number of university students having felt that fascination for teaching. In fact, in the FoPD17 edition, there were 99 volunteer students who performed their experiments for people of all ages and conditions and had been captivated by these fascinating experiments with plants. Do you also want to be caught by the fascination of experimenting with plants? Well, turn the page and experiment with us!

Fascinating molecules

INTRODUCTION "Almost all aspects of life are engineered at the molecular level, and without understanding molecules we can only have a very sketchy understanding of life itself". [about DNA] Francis Crick

Living organisms are formed by organic molecules, which are those molecules constituted by carbon atoms that covalently join together forming a skeleton and which can also contain chemical groups including mainly hydrogen, oxygen, nitrogen, phosphorus and sulfur atoms. The properties and functionality of organic molecules depend largely on their size (determined primarily by the number of carbon atoms), their spatial configuration (molecule shape), and the functional groups linked to the carbon atoms.

Living things, including plants, are capable of producing a large number of organic molecules that contribute to their growth and have very diverse functions. Many of these molecules are included in one of the following four main groups: carbohydrates, lipids, proteins and nucleic acids. Carbohydrates can be single molecules (monosaccharides), disaccharides two linked monosaccharides) and polysaccharides. The first two are used by cells as a source of energy and components of other compounds. A clear example of this is glucose. The disaccharide

Romina Martínez Rubio & María Luz Centeno Martín

sucrose is the form in which sugars are transported in plants. Polysaccharides have a structural role when they are part of cellular components —for example, the cellulose and hemicelluloses in the cell walls of plant cells— or are energy reserve molecules and carbon atoms that the cell hydrolyses and uses when needed (starch, see chapter 4). Some plant lipids also represent forms of cellular energy storage, such is the case of triglycerides (fats and oils), and others are major components of biological membranes (phospholipids and sphingolipids), so the function of the latter would be structural. Others play special roles, such as cuticle waxes, which help reduce water loss in exposed parts of the plant. Also the carotenoids, pigments that participate in the capture of light in photosynthesis, belong to a group of lipids. Proteins are made up of amino acids, their simplest molecular subunits (Figure 1.1), and they are the most versatile macromolecules in cells. They are involved in almost all metabolic processes, since most enzymes, molecules that accelerate reactions in organisms, are proteins. They serve as structural components, such is the case of tubulin and actin, two constituent proteins of the cytoskeleton. There are also reserve proteins such as glutenins and gliadins that store cereal seeds. Others participate in cell transport, as, for example, the membrane channels, or have regulatory functions (activators and repressors of gene expression, signal receptor proteins, etc.). In fact, the set of proteins that each type of cell possesses largely determines its morphology and its functions. Nucleic acids are, like proteins, large and complex molecules. They can be of two

types: deoxyribonucleic acid or DNA, and ribonucleic acid, RNA. The first represents the hereditary material of the cells and the molecular basis of the genes, which contain the instructions for the synthesis of all proteins. The RNA transmits the information of the DNA from the nucleus to the cellular cytoplasm, where the protein synthesis takes place, process in which the RNA participates directly. Some RNAs can even act as biological catalysts (ribozymes).

Figure 1.1. Chemical structure of the amino acid serine. Balls correspond to carbon atoms (black), oxygen (red), nitrogen (blue) and hydrogen (gray).

In addition to these four groups, plants synthesize other molecules, usually smaller in size, but with equally important functions. Among these, secondary metabolites can be highlighted, so called because they do not seem to have direct functions in the development of plants, and plant hormones. Many of the former have ecological functions, so that they protect plants against herbivores and pathogenic microorganisms, attract pollinating insects, or try to avoid colonization of the environment by competing species of the one that produces them. Plant hormones are molecules that regulate all the development processes that occur in the plant throughout its life cycle, as well as

1. Fascinating molecules

many of the plant's responses to different stressors. In this chapter two experiments are presented. The first involves a type of macromolecule, DNA, which can be

extracted from different plant materials by applying a very simple method. The second one works with the small, minor regulatory molecule ethylene, the plant hormone that regulates the ripening of many fruits.

Romina Martínez Rubio & María Luz Centeno Martín

1.1. OBTAINING VISIBLE DNA EXTRACTION OF DNA

SUMMARY DNA (deoxyribonucleic acid) is the molecule where the genetic information of every living things resides. The nucleotide sequence that forms the DNA of each organism is unique and characteristic of it. This is why the extraction and subsequent analysis of the DNA can be applied to determine the paternity of an individual, identify a criminal from samples collected at the scene of the crime, or know the vine varieties from which a wine was made. These are just a few examples of its many applications. This chapter shows a simple method of extracting pea DNA and observing it with the naked eye, without the need for a microscope or the use of sophisticated laboratory techniques.

INTRODUCTION DNA is the biological molecule that contains the genetic information of every organism, including plants. Don’t despise them for being those green "things" devoid of feelings... They are living things just as us! The DNA is considered the inheritance molecule since the cells are able to replicate their DNA to give rise to two identical copies that are then distributed equally between two daughter cells during the cell division process. Thanks to the replication of the molecule and cell division, a cell is able to transmit the genetic information stored in its DNA to its descendants. Prokaryotic cells, like bacteria, have a circular DNA found in the cytoplasm. In

animal eukaryotic cells, DNA appears in two locations: a) nuclear DNA, which is made up of different numbers of linear molecules (depending on the species) associated with proteins, forming chromosomes, and b) the DNA we find in the mitochondria, represented by hundreds of copies of circular DNA, very similar to that of bacteria. Plant eukaryotic cells, in addition to having nuclear and mitochondrial DNA, have DNA in the chloroplasts, which is also made up of abundant circular copies. According to its biochemical composition, DNA is a polynucleotide, that is, a polymer consisting of units called nucleotides. Each nucleotide is composed of a nitrogen base, which can be adenine, thymine, cytosine or guanine, the deoxyribose monosaccharide and phosphoric acid. Each nucleotide binds to the next one through a phosphodiester bond. As the phosphate groups in solution are dissociated and have a negative charge, the DNA molecule as a whole has a negative charge, and therefore an acidic character, as its name reflects. The nucleotide sequence of the DNA of each living thing is characteristic and unique, which allows its identification through the analysis of the molecule. In this experiment, we will extract DNA using as our starting material different parts of plants and applying extraction method based on the chemical properties of their molecule.

1. Fascinating molecules

Figure 1.2. Materials used in the realization of the experiment.

The DNA discovering In 1952, James D. Watson and Francis Crick proposed a model to explain the secondary structure of DNA. For this, they based on the results obtained by other researches, such as Rosalind Franklin, Erwin Chargaff and Maurice Wilkins. The model they proposed was denominated since then the DNA double helix. According to the model, the molecule is formed by two complementary polynucleotide chains, antiparallel disposed. The nitrogenous bases on the two strains dispose towards the inner part of the structure, pair up and held together by hydrogen bonds. Phosphate groups are located outward.

MATERIALS To perform the experiment we will need the following materials (Figure 1.2): • 250 grams of fresh or frozen peas. It can also be made with a large onion, with 100 grams of banana, etc. • Pineapple juice. • Glass containers. • Dishwashing liquid. • A blender. • Water and salt. • A strainer. • A graduated cylinder. • A spoon. • A 96% alcohol solution, very cold.

Romina Martínez Rubio & María Luz Centeno Martín

EXPERIMENTAL METHOD To extract the DNA, the peas are placed in a glass container and completely covered with water (if a larger vegetable is used, such as an onion, it’s better to chop it into smaller pieces beforehand). Then the peas and water are blended in the blender. Two tablespoons of dishwashing liquid and a teaspoon of salt (about 3 grams) are added and the mixture is stirred vigorously with a spoon for about 5 minutes. Then the mixture is left to stand for 15 minutes. After that, the mixture is poured through a strainer over a glass container to separate the solids from the liquids. Leave the liquids in a glass container. For the rest of the experiment, use only a portion of the strained mixture. About 50 millilitres of the strained mixture are put into a graduated cylinder and then three spoonsful (15 millilitres) of pineapple juice are poured over it. The tube is covered with the hand and everything is swirled together. It is let to stand for 15 minutes. Any foam that forms at the top is carefully removed with a paper towel.

Finally, an equal amount of very cold ethanol as the amount of pea-pineapple juice mixture already in the graduated cylinder is slowly added. The alcohol is poured so that it slides down the side of the graduate cylinder and does not mix with the pea-pineapple juice mixture. Due to its lower density, the alcohol phase (containing cell debris and extracted compounds from the plant material) should remain on top of the pea-pineapple juice mixture so that there are two layers in the graduate cylinder. The graduate cylinder is let to stand for another 5 minutes. During that time, white filaments will begin to appear between the two layers and rise into the alcohol layer. It’s the pea DNA! (Figure 1.3).

WHAT HAVE WE LEARNED? To extract DNA, the first thing that is done is to break the tissues, which is why the plant material is crushed in the blender. Adding the dishwashing soap then disorganises the cell membranes, including the plasma and nuclear membranes. In this way, the DNA contained in the cells is

Figure 1.3. Result of the experiment. The “balls” or white lumps that appear at the interface of the alcohol and pea-pineapple juice mixture and then ascend in the alcohol layer are the strands of DNA.