Clone Guide #
Introduction #
This guide focuses on the context of Amsterdam, with its old town full of trees and a lot of green spaces and parks. Moving to the Netherlands with a naïve way of looking at things, it feels like nothing is left to chance, in particular in relation to water and plants. The flat topography entices the naïve and highly subjective notion of everything being controlled and shaped by people, enabling infrastructure to be built in industrial shapes: rectangles and straight lines. Riding a train through this country, disregarding city and mobility infrastructure, one sees water infrastructure, rectangular farms, and almost none to sole thin and young forests. And even these trees look orderly placed.
On Cloning #
The term “cloning” is used intentional imprecise in this guide to allow space for interpretation. Technically speaking, the cloning of plants is an asexual propagation of an existing plant that then contains the same DNA. In the sexual propagation, two sets of chromosomes of at best two different plants are brought merged into a new full set of DNA. The vast majority of plants does this with pollination through wind or other external help such as bees, to create seeds which then are distributed again.
While there are plants that use asexual propagation without humans as a driver, propagation techniques are applied in nearly every plant in the industry and agriculture. This is due to many benefits like faster, almost completely automated production and the ability to ensure controlled similar plant properties from the original plant. This enables to do research to be much more precise and guaranteed crop quality that all are ready to be harvested at the same time.
Cloning techniques do not have to be high-tech at all with thousands of year old techniques of sticking cuttings into soil or crafting that is applied for many fruit trees.
Since the 1960s, the field of in-vitro tissue culture started to rise, which this guide is focused on. In this method, tissue from specific parts of a plant are sent into an induction medium containing all the nutrients, namely minerals, sugar, vitamins, and hormones to start new plant cells. After a few days of growth, small platelets can already be multiplied to a nearly infinite number of time. Other advantages over seeds are that the DNA is identical but not ambiguous and that, if setup correctly, there is less error than with seeds.
Structure #
This guide is a starting point to clone trees, their bodies and their stories. The first part contains a hands-on DIY guide to micro-propagation (a common industrial reproduction of plants), how to find resources and build a lab. Furthermore it will be explained how to prepare test tubes containing the medium which includes all the nutrients (minerals, vitamins, sugar) and hormones, as well as how to excise plant tissue which, placed in the medium and the right conditions, starts to grow. The guide’s second part contains tree stories from a fictional reality set within the city of Amsterdam, an attempt to propagate the subjectivity of a more-than-human matter. The stories zero in on the tree’s relationship(s) with humans by and through the industry, planing, maintenance, and co-dependency. These stories are written in an accessible and informative format which, purposefully, does not seek to anthropomorphise.
The Guide’s Limits #
There are a lot of great guides that are already written, much more extensive, with much more knowledge, depth and written by educated and respected in this field. The intention of the guide is not to provide accurate knowledge but an introduction and debate of the practice, intentionally incomplete. This guide is written by an amateur, which sets the limit of what you can (scientifically) learn from it. Cloning trees is not easy for amateurs, but possible. Do not expect immediate results, particularly not after reading this guide alone. It takes a while and there may be many small interferences. Especially, preventing contamination is a basic requirement but also requires many experiments and much experience. Even in the industry, a significant numbers of contaminations occur. Working with a living material is rarely exact and vast amounts of what is happening remains unknown. Lastly, respectfulness is important. Tissue culture is a whole scientific discipline and industry with years of knowledge and experience based on plant research. It is thus important to acknowledge professionals shaping and working in the field. Please repsect not only people, their life and professions but also the (more-than-human) world around you. Our actions affect our vicinity. Especially more-than-human beings that we have little awareness of and easily slip our attention and sight and their stories that remain untold. Even if it might not seem alive, it is.
Format #
This guide was part of the master program Studio for Immediate Spaces at Sandberg Institute. The intention is to be accessible and give an overview to the practice of tree cloning. Therefore, the guide is incomplete and does not intend to provide the accuracy of a scientific one.
Instead of creating a physical publication that is restrictive in the access, the digital format is a choice with the hope of being more accessible to people out- and inside of an art context. The incompleteness allows the guide to be alive, corrected and new knowledge allowed to enter. At the same time, alive means that it required electric and human labour to be fed to keep it alive.
Personal Position #
It feels off writing and experimenting with trees in a world that is on fire. I came to The Netherlands from a place that historically feels politically even more isolated from the reality happening outside. What happens to the trees of Amsterdam does not appear with any urgency in relation what happens in other places in the world. The ecological, economic, and political effects of the climate crisis are vividly present in my life but the consequences are minor compared to other places and people on the this planed minor. In my close surrounding, there are many people who are deeply worried about the future to come but at the same time are disengaged.
This guide is a trial for exploring possibilities to battle disengagement with physically and mentally. Looking close at the trees of Amsterdam might be non-sense but could open ways of engaging with a more-than-human-world and earning a sensitivity that can be applied more universally then being tied to one place.
It would be easy to write a fatalistic piece. But instead of falling into a cynical mode of “knowing better” and turning passive, I would rather be naïve but have hope. Otherwise, it would not be worth it anyway.
It is a designers pattern to see the world and politics through Artifacts, which In my opinion can be problematic and I am not exempt from it.
Cloning Tree Bodies #
Resources and Material #
The Netherlands are a great place to source hardware, chemicals and knowledge. Leading agricultural research institutions, specialty hardware stores and biochemical suppliers are historically and to this date mostly located in this country. This is due to the Dutch colonial history and the associated emerging research for new plants which were then commodified and commercialised. It is thus important to remember that tools and techniques can be repurposed but their, shape, usage, practice, and thus meaning carries a history deeply rooted in colonial histories of pain and injustice. Technology and design is inevitably political and is shaped by people with power. Juxtapose own thinking, abilities and possibilities with actions and responsibilities.
Nevertheless, since the cultivation of cannabis and mushrooms, containing THC and Psilocybin respectively, was made illegal, enthusiast and (hobby) researchers work in the hidden, largely at home. Therefore, knowledge and practice are often simplified with accessible tools and exchanged online, alternative to professional research or commercial labs. If you look for ways to work sterile (outside of a lab), take a look at the fungi people. Micro-propagation might need extra arrangements but it is a good source of inspiration. If you need tools to grow, the cannabis people are the ones feeding your creativity.
Knowledge #
One aim of this publication is to make knowledge and skills accessible and thus enabling a critical and nuanced debate around the cultivation of plants. Obstacles frequently occur when this knowledge is saveguarded by ownership as well as when limited accessibility in terms of language requirement and prior knowledge(s) become challenging to tackle. It is thus important to share resources as well as the knoweldge they transmit to increase accessibility.
Books #
Plants from Test Tubes : An Introduction to Micropropogation #
This book is comprehensive and easy to understand for beginners. It covers fundamentals ranging from the history of micropropagation, basic chemistry, equipment and laboratory design to preparing stocks and media, explant selection, preparing explants, grow rooms, transplant preparation, troubleshooting, and even gives tips on how to start a business. Additionally, it provides many easy to follow protocols with references to original sources. This book is my personal recommendation. Most technicalities that you can read in this guide can be read there in much greater detail.
Other Books #
Following recommendations are by big parts unreviewed by me:
- Plant Tissue Culture: A Home-Based Guide: How to Practice Plant Tissue Culture on a Budget, by Edward E Johnson
- Protocols for micropropagation of woody trees and fruits, Shri Mohan, Jain
- The Plant Propagator’s Bible: A Step-by-Step Guide to Propagating Every Plant in Your Garden, by Miranda Smith
- The Reference Manual of Woody Plant Propagation: From Seed to Tissue Culture, by Michael A. Dirr
Scientific Articles #
Because each plant requires an individual approach to micro-propagation, it hence also requires a specific protocol for each plant to follow. The quality of these scientific protocols varies a lot but most often, they are hard to access, hidden in scientific publications and come with a heavy price tag. For people who approach this as a DIY practice and do not make excessive profit from it, paying these prices is not reasonable and often further imposes limitations on accessibility.
Here, it is important to mention the effort of many (internet) activists advocating for copyrights for the common public. One prominent figures was the internet and copyright activist Aaron Swartz who explicitly spoke against restrictive centralised, privatised digital publishing sites. As an activist, he downloaded papers from the MIT computer network and later published them freely available on the internet (Gerstein 2011) (MIT Police Incident Report 2011). As part of the Open Access Movement he stated in his Guerilla Open Access Manifesto:
The world’s entire scientific […] heritage […] is increasingly being digitized and locked up by a handful of private corporations[…] The Open Access Movement has fought valiantly to ensure that scientists do not sign their copyrights away but instead ensure their work is published on the Internet, under terms that allow anyone to access it. (Swartz 2008)
Swartz committed suicide shortly after being arrested and put under immense pressure by being threatened with heavy legal charges to long imprisonment. One of his many legacies is the base for a lot of online “piracy” websites that besides books, share scientific papers thus making them accessible to everyone with free internet access.
Thus, if you can afford, pay the journals and published papers and, quite literally pay, respect to the works of many scientists that enabled you to do the things you want to do. I do not want to persuade you to commit copyright felonies but if you do not have the means, these are good alternatives.
Additional Sources of Knowledge #
The practice of micro-propagation is experience based, which requires many experiments with failures and productive frustration. But there are also many other places where you can learn from people with a lot of experience, who can all add something with their specific way of doing it. A good place to start is to get in contact with people from existing institutions such as universities and commercial organisations. However, beware that the interest of non-professionals can often be overlooked or misunderstood. It is worth to look for other places where people are open to exploration, DIY, and amateurism, often combined with political fields, such as Open Wetlabs and Bio-Hacker spaces.
Open Wetlabs #
- Waag, Amsterdam
- MadLab, Manchester
- Open Biolab, Graz
- La Paillasse, Paris
- DIYBio Tours
- Brmlab Biolab, Prague
- Biotehna, Ljubljana
- Hackteria, Zurich
- DIYBio, Groningen (looks inactive)
- Biotinkering, Berlin (looks inactive)
- London, Biohackers (looks inactive)
Hardware #
As mentioned above, the partly decriminalised growth of cannabis containing THC for personal, non-commercial use enables the easy accesibility to as well as availability of professional-appearing equipment specific to the Netherlands. Vast offerings of growing equipment are handily available compared to Switzerland, where exclusively THC low plants are legal. Due to the (semi-)illegality in many countries, equipment (and knowledge) commonly comes in a form that can be applied hidden at home and shared anonymously but accessible online. Furthermore, many tools can be bought in household stores, which are affordable and allow to buy smaller quantities. Specific tools can be substituted with household articles and are sometimes even produced for both, professional and homeware, applications, sold in separate stores. For example: Professional, long forceps are identical to the ones used to swirl pasta.
Chemicals #
The more specific the chemicals become for tissue culture, the harder it becomes to source them. The majority of the plant biochemistry stores exclusively sell to commercial or research institutions. It requires creativity to stretch the term research in combination with trust built through previous contacts. Once esatblished in the system, access stays unlimited and prices are comparably cheap but quantities can be outside of the home DIY scale. Thus, it is a good practice to buy small amounts and sometimes consider other suppliers. For example, it might be cheaper to buy 5 litres of bleach but storage and disposal is not in harmony of effort and valuing time, work, and environmental resources. From personal experience, Duchefa Biochemie in Haarlem, one of the biggest suppliers worldwide according to their own claims, has en extensive inventory of biochemicals and tools, are forthcoming and helpful, and allow picking up orders.
Build a Lab #
Before diving into heavy investment for your own, it can be an option to ask for access to existing infrastructure. It is handy to work in a space that was purposefully designed to be used as a lab and contains all high-end equipment. On the downside, getting access granted can be difficult and can be limiting in the time it is available for use and the extra time it requires to get to know a lab and their practice(s). Done the right way, building your own lab can be a fun activity that brings you a deeper understanding of the material. It however requires financial investment, time and frustration, and is a never ending task.
Sterile Conditions #
Before precision, working sterile is actually the most important and one of the most difficult conditions that needs to be achieved. Even in the most “artificial”, the environment around, on, and within us is alive. The air is full of fungi spores (likely mold), bacteria, and viruses. Their way to proliferate is to multiply or grow and spread quickly, suppressing everything that is weaker than them. Furthermore, our body hosts a huge amount of these living beings that we codependent on. Likewise these species live on and in plants, which is one reason why micropropagation is a popular choice in the industry because the minimal use of tissue is less likely to contain these “contaminants” and can be sanitised much easier. In the beginning, before their immune system exists, the explants are extra fragile to all of these contaminants. Working under sterile conditions is therefore necessary so that the air, every surface and every touch cannot destroy the project before it even starts. Wearing masks and gloves, frequently sanitising hands, surfaces and tools helps. But two tools are inevitably helpful and fundamental for making sterile conditions possible: an autoclave and a laminar flow hood. While the autoclave is inexpensive and accessible, laminar flow hoods are not and it cost-efficient ways have to be found.
Autoclave #
There are many different types of autoclaves but the most popular and at the same time accessible one, is a pressure cooker. While professional autoclaves can vary in function, have a bigger volume, and can work automatically with a timer and water supply, a pressure cooker does the same and is inexpensive. When on full pressure, a pressure cooker is at 121 °C. Protocols require autoclaving for 20 to 30 minutes, meaning leaving if on full pressure for that time. During that time, everything that is alive is killed by the heat. That comes in handy if the gelling agent (such as agar) needs to be heated in order to liquefy and become clear.
Be aware that not every material can be autoclaved because it could melt or burn. Pay extra attention when it comes to plastic and paper and do your research. Test tubes would likely melt by those temperatures and need to be bought sterile. Flammable material (such as paper) could be placed in an autoclave-save plastic bag. Be sure to follow the recommended use by the pressure cooker producer in particular on how much of the volume is filled.
Not every type of glass can be autoclaved. Therefore it is wisely to use glass that can withstand the autoclave temperatures.
Make sure not to close any container since matter expands with head and could explode and damage the cooker, which then would be dangerous. It is recommended to closely close containers by tightly wrapping aluminium foil over the openings to cover them from condensed water and other things falling in.
Once you sterilised the content of autoclave, you need to make sure it stays sterile with the help of the laminar flow hood.
Laminar Flow Hood #
A laminar flow hood is a device that blows particle filtered air in an intensity that the airflow is straight and without any turbulence. A laminar airflow. While a clean working area is essential for preventing contamination, it is impossible to achieve an absolute sterile room, in particular with limited resources. By turning on and correctly tuning a laminar flow hood, it can be ensured that the few centimetres of space in the front area is nearly free from particles and therefore contaminants. This tool provides an immense simplification of keeping up sterile conditions makes this practice accessible for non-professional settings.
But a laminar flow hood requires investment. Affordable options from fungi equipment suppliers or Aliexpress/Alibaba are available but quickly exceed 400 euros, while it is not difficult to build one yourself. Thanks to the fungi community, again, there are a lot of tutorials on YouTube, which provide step-by step instructions. Summarising these, a box with one hole of the size of the HEPA filter in front and one for the fan (not directly facing the filter, on the side or from the top) has to be made that is air tight. This can be custom made with wood or with ready made and closed plastic boxes, while the latter would limit you in the size that is needed. For the fan, it is important to buy one that is strong enough and has an adjustable, continuously speed controller that at best is integrated but can also be separate. At best, it is a brushless motor. There are offers for fans for indoor growing at growing shops. HEPA (High Efficiency Particulate Air filters) have classes for their filter strengths which H14 should be sufficient for the work of micro-propagation. There is a big price span from 60 to 300 euros, depending on the size and producers. Since electronic producers sell home air purifiers, small HEPA filters are available at an affordable price on every day online markets. When watching YouTube tutorials, keep in mind that compared to the work with fungi, the work with tissue culture needs more equipment that needs to remain sterile and therefore more space is needed.
Precision Scale #
Essential to measure chemicals for the preparation of the plant medium is a scale that has a precision of 1 mg because protocols require it. Affordable ones are usually limited to a precision of 10 or 3 mg but technology is getting smaller and cheaper. Nevertheless, the specs have to be carefully read and not all producers can deliver what they promise, which requires extra caution. A more promising way to find a suitable scale, is to regularly check online billboards for second hand scales, which appear from time to time. Known scale brands are Kern or Mettler, which might be sold when a lab is closed.
Grow Box #
Getting a box to grow is straightforward. For the propagation of plants, there are cheap boxes with transparent plastic available that can enclose explants once they are placed in the test tubes. In there, a heating mat is a cheap and energy efficient method to heat the box. There are plenty of power plugs available that are attached to a thermostat that turns power off and on if the desired temperature is reached. The same power plug should include a timer option for the light. The majority of the protocols require the explants to receive 16 hours of artificial sunlight which can be provided by full spectrum lights. It is worth to check the protocols to figure out which light range and intensity the plants require. Specific LED lights are sufficient for common cases and also save energy. If the plants do not receive any sunlight, the timer on the lights can be turned on in the night to use the cheaper electricity prices. But just as humans, the plant cells need a time without light to rest. This grow box can later be used to acclimate the grown plantlets to soil and air.
Material List #
| Name | Category |
|---|---|
| HEPA Laminar Flow Hood | Tool |
| Pressure Cooker | Tool |
| Scalpel | Tool |
| Forceps | Tool |
| Microwave | Tool |
| Mechanic Pipette (100µl and 1000µl) | Tool |
| PH Strips | Tool |
| Beakers (400ml and autoclavable) | Tool |
| Face Masks | Tool |
| Grow Box (With full spectrum lights, heating mat, and thermostat and timer power plug) | Tool |
| Precision Scale (1mg accuracy) | Tool |
| Kitchen Scale | Tool |
| Scissors | Tool |
| Disinfect Dispenser (Wash Bottle or Spray) | Tool |
| Hot Plate | Tool |
| Lighter | Tool |
| Ethanol / Isopropyl | Consumable |
| Test Tubes (sterile) | Consumable |
| Bleach 5% sodium hypochlorite (or higher to dilute) | Consumable |
| Distilled Water | Consumable |
| Paper Towels | Consumable |
| Disposable Gloves | Consumable |
| Disposable Plastic Containers | Consumable |
| Autoclavable Glass Containers | Consumable |
| Face Masks | Consumable |
| Aluminium Foil | Consumable |
| Painter’s tape | Consumable |
| Murashige & Skoog Basic Medium (MS) | Chemical |
| NaOH – Natriumhydroxid | Chemical |
| IBA – Indole-3-butyric | Chemical |
| BAP – 6‑Benzylaminopurin | Chemical |
| NAA – 1‑Naphthaleneacetic acid | Chemical |
| HCl – Hydrogen chloride | Chemical |
| Agar | Chemical |
| Activated Charcoal | Chemical |
| Sucrose | Chemical |
Preparation & Protocol Example #
While each plant type requires an individual procedure and protocol, the process of micro-propagation is similar. In the interest of simplicity, this guide demonstrates a protocol for the micro-propagation of a fig tree.
Principle #
Chemicals travel through a plant, sometimes created by the plant itself, taken up from the soil, cooperating fungi and changed through photosynthesis, and are deployed to the places of new growth, commonly the tips of a twig or in the roots. The specific tissue that is located there is called the meristem and contains all cells (undifferentiated cells) to produce into every other type of cells of a plant. The hormones (called growth regulator) are a cellular messenger that defines in which way and which type the cells should grow. Other than animals, plants do not have a centralised organ system and everything that happens within a plant is reproduced in the chemical mixture of the container. To put micro-propagation or in-vitro tissue culture into simplified terms, specific tissue of a plant is put into a media in a sterile container that contains all the nutrients (such as minerals, sugar, and vitamins) and growth regulators to grow and eventually root.
Medium Base #
While the simplified explanation of micro-propagation sounds easy, it is an enormous field of scientific research that cannot be picked up swiftly. Knowing, sourcing, and possessing/storing all the right chemicals is not feasible for amateurs in the focus of this guide. Luckily, there are many applicants who cannot bother to dive that deep into material sourcing and pre-mixed base media that exists. One of the earliest and prominent ones is the Murashige & Skoog Medium (MS) that was developed in 1962 by plant scientists Toshio Murashige and Folke Skoog and is the basis for this protocol example. This medium can be inexpensively bought at many chemical online shops – even ones that are not specialised in tissue culture. There are a couple of variations in strength and the in- or exclusion of vitamins. Choose the regular one with the vitamins for this example.
Groth Regulators #
Most common categories of growth regulators are Auxins and Cytokinins. “Auxins are phytohormones that influence cell enlargement, root initiation, and adventitious bud formation”. And, “Cytokinins are growth regulators that are required in tissue culture media for cell division, shoot multiplication, and axillary bud proliferation”. (Kyte 2013) These two components need to be combined in a ratio that lets the cell grow in the desired way. The growth-regulators that are used in this example (Auxin: IBA and NAA, Cytokinins: BAP ) are common ones that are appearing in various protocols.
Gelling Agent #
While there are ways of doing micro-propagation with liquid culture, this guide focuses on propagation in a gel, agar respectively. What is repeated in this guide multiple times, is to check the specific protocols. Nevertheless and compared to the other components, the gelling agent could be substituted. It can be said that the more purified and the clearer the gel is, the better. Contaminations are more likely to be spotted in clear gel. For that, it is important to get specifications of the gel strength of both, the original and the substitution and calculate a new composition. At least, this was working out well for my experiments.
Creating Stocks #
Because the growth regulators are used in small doses (in this case 2 mg and 0.2 mg), even a precision scale is not precise enough. To cope with that, these chemicals are diluted in distilled water and then later pipetted with mechanical pipettes into the medium. To simplify calculations, 25 mg of either Auxins or Cytokinins are diluted in 250 ml of distilled water. To add 1 mg of the growth-regulator 10 ml of the diluted stock has to be added to the medium.
To do that, 25 mg of the chemicals have to be added to a 400 ml beaker. In the case of Cytokinins, “1 M HCl solution one drop at a time until the BAP is dissolved” has to be added (Kyte 2013). To help with dissolving, head can be applied to the beaker. Once dissolved, 250 ml of distilled water can be added, tightly closed, labeled and stored in a fridge. With Auxins, it is the same approach that differs with the use of 1 M NaOH or KOH to dissolve the chrystals and there is no need to heat.
Selecting Cuttings #
For the example of the fig tree, buds of new growth are cut. In general, it is recommended to do cutting of young plants and in late winter around January and February. On one hand, there are less contamination in young plants and “dormant” buds in winter are more likely to react well on stimulation through the medium. They are ready and prepared to grow in spring. A few centimetres around the bud should be cut off and put into a container with moisturised paper towels to prevent them from drying.
Prepare Cuttings #
Since not only surfaces and the air is full of contamination, the cuttings themselves contain bacteria, fungi, and viruses which need to be sterilised to not take over the plantlet in the test tube. In the case of this fig propagation protocol, all excess plant material, everything woody and brown is removed, even a few outer shells of the bud can be cut away with a scalpel. The prepared buds are rinsed in running tap water for 45 minutes. Later the buds are sterilised in 5% bleach, in this case sodium hypochlorite. Be aware: to dilute more potent bleach with distilled water, always fill up the water with the bleach and not the other way around. Otherwise, the solution will boil up and make a big and dangerous mess. The cuttings can be wrapped in a cheese cloth and put into a test tube to ensure that all the surface of the buds is covered by bleach. Timing is important, in order to not destroy the plant tissue. After five minutes the buds have to be removed from the bleach and placed in one of the three separate water baths where they can stay until the last ethanol bath before the final cutting to be placed in the test tubes.
Prepare Medium #
Depending on how much medium is needed, it is a good practice to calculate and write down the exact measurements. Calculating the milligrams of growth regulators into microlitres can be confusing. In the case of a stock solution of 25 mg in 250 ml of water and a desired amount of 0.2 mg, the formula 250 ml / 25 mg * 0.2 mg gives you the desired result of 2 ml that are needed to put into the medium. To clear even more confusion in this stressful moment, that means 20 pumps with a 100 µl mechanical pipette. All components have to be put into an autoclave proof beaker with the correct amount of distilled water. A microwave is a handy tool to bring the medium to temperature and melt the gelling agent. Make sure not to cover the beaker while microwaving and regularly stop to stir the mixture until clear and dissolved. While the medium is hot, it should be placed in the autoclave with all the heat proof tools that are used in the later process. Covering the containers with aluminium foil prevents water and other things to enter and change the composition of any liquids. Autoclave for 20 minutes on full pressure.
Prepare Sterile Desk #
While autoclaving, the sterile work station in front of the laminar flow hood can be prepared. The laminar flow hood needs to be running for at least 15 minutes before use to ensure particles that were inside of the filter were blown out. In order to achieve a laminar flow of the air without turbulences, the regulator has to be set so that a flame (i.e. from a lighter) bends in a 45 percent angle and does not flicker. To simplify sterilisation of the surface, which needs to be disinfected with 70% ethanol/isopropyl first, it should be covered in aluminum foil, attached to the table with masking tape and then sterilised again. Disinfect everything that is in front of the laminar flow hood with 70% of alcohol. Wear plastic gloves that are desinfected every few seconds before touching anything. If they get dirty, discard them and use new ones. Once the autoclave is done, the content should be immediately placed in front of the laminar flow hood wearing freshly disinfected gloves before touching the content directly. The test tubes should be placed in front of the laminar flow hood in a rack and disinfected throughout. Everything should be in place and prepared that the tubes are opened as shortly as possible. After disinfecting the working hands and the tubes once again , open them up all at once, fill them as quickly as possible with the, until this moment, liquid and hot medium from the autoclave and fill them with enough headspace for the later placed plant. Now the medium has to cool down and solidify for the next steps.
Excision #
Every process that requires multiple steps including sterilising hands, surfaces, and tools, should be a routine that can be played through beforehand. As a principle, try not to touch any surface and keep your fingers as far away as possible from any material. Long forceps are a great help with this. In the cutting of the plant tissue, the meristem should be as close to the medium as possible. One way of ensuring that, is by cutting out the meristem itself. Špela Petrič showed me how to do that with a cosmetic syringe, which can cut away the inner leaves and shells of the bud and eventually point through the tissue and lift the round meristem dome with the inner of the needle tip.
This is not always needed. It could work to cut away all the excess tissue and halving what is left of the bud. This way, the meristem is close enough to the medium in order to grow.
Grow Box #
In the case of the figs, the test tubes have to be placed upside (at best in a rack) in the grow box without directly touching the heating mat. The lights have to be set on 16 h a day and the temperature regulated between 24°C and 25°C.
Full Protocol #
This protocol is a simplified version from the book “Plants from Test Tubes” for Ficus carica.
EXPLANT Actively growing apical shoots. Milky latex will be produced from fresh cuttings, but the use of antioxidants will decrease loss from polyphenolics. Keeping the new explants in the dark for one week after initial culture has also shown some benefit.
TREATMENT Wash explants thoroughly under running tap water for 45 minutes followed by immersion in 2:10 bleach for 5 minutes. Rinse in sterile water 3 times. Immerse in 70% ethanol for 30 seconds and rinse with a sterile antioxidant solution of 0.01% L‑ascorbic acid.
MEDIA MS medium with 2 mg/liter BAP and 0.2 mg/liter NAA for Stages I and II. For rooting, use half-strength, liquid MS medium with 2 mg/liter IBA and 0.2% activated charcoal.
Ficus media
Compound Stages I & II mg/liter Stage III mg/liter MS salts 4,628 2,314 NAA 0.2 – BAP 2.0 – IBA – 2.0 Sucrose 30,000 30,000 Agar 8,000 – Charcoal – 2,000 pH 5.7
LIGHT 150 – 200 f.c. from cool-white fluorescent light for 16 hours light/8 hours dark.
TEMPERATURE 24°C – 25°C (75°F – 77°F).
(Kyte 2013)
Cloning Tree Stories #
Manufactured Elm #
If one tree can be seen as the tree of Amsterdam, it is the Elm. In 2005 Amsterdam declared itself the Elm City of Europe as the vast majority of trees in Amsterdam are Elms and thus part of the city’s landscape heritage. Before, when the citizens of Amsterdam started to plant trees next to the canals, a lot of linden were planted which are considered majestic but sensitive. The elm, on the other hand, is low-maintenance and adjusts well to the city’s climate by its upright growing branches, saving space to make room for the growing human living spaces. But what makes this tree even more Dutch than its cultural meaning is its engineering. In 1921 two Dutch phytopathologists identified a fungi now known as the Dutch Elm Disease, as it particularly devastates elms. While the fungi are catered by the bark beetle that helps it to move and stay alive, the elms in Amsterdam are gradually replaced by a new type of elm that was designed by the Dorschkamp Research Institute for Forestry & Landscape Planning, Wageningen. This type, the Columella, is an accidental cross between the Plantyn elm, which was the first engineered attempt to make a resistant hybrid, and the field elm. This cross-pollination happened without direct human interference. But due to its extreme resistance to the Dutch Elm Disease, the Columella was commercialized nevertheless. Since 1989, the tree has been commercially available and planted all over Amsterdam. This partly artifically, partly naturally engineered tree replaced the original rotted elm.
40 Year Old Tree #
The target lifespan of trees is set at 70 years before they have to be replaced by the municipality of Amsterdam. In reality, the majority of trees needs to be replaced when the trees are 40 years old, around 20 years after they have been planted. Since Amsterdam is a both windy and dense city, security is one of the municipality’s main concerns. Therefore, the city surveils and regularly assesses one third of the 300,000 tree “assets” in its responsibility within a year before they may be a potential danger to people and their possession in public space. In addition, the Bomen department is obliged to react and cut down dangerous trees within 48 hours. The efforts to keep track of every single tree are tremendous. Previously “[e]ach of the – at the time – 14 city districts had its own manual and policy frameworks” (van het Hof 2019) and registry in their own way, seriousness and precision (Reeskamp 2024). This brings the potential for serious issues with trees that are not recorded and have an unknown health state. As this issue exceeded human scale, the municipality is deploying drone search flights equipped with Lidar 3D laser scanning. In the course of the implementation of the Puccinimethode, among manual for public space, offices, and processes, the registry of all trees in their responsibility was centralised. The Puccinimethode is a design management manual for the implementation of surfaces by the municipality of Amsterdam that contains the “Handboek Groen” (green hand book), a handbook that defines the city’s “ecosystem surface”. As beautiful as the manual’s name sounds, it is, just as planting a fig tree in the Netherlands, another attempt to bring the Mediterranean into the dull Dutch environment. One of the initial meetings about the later coined Puccinimethode was too early in the day to serve Bitterballen, which prevented the method from being named “Bitterballenmethode”, the less euphonic name of the the dutch stew thickened, breaded and deep-fried roux meet balls, representative for the dutch culinary maturity, served as snacks for the meeting. Instead, and for the better ring, chocolate bonbons by the nearby Puccini Bomboni chocolatier were served which led to borrow the name for the method as an inspiration for its perfection to be reached on the streets of Amsterdam, the municipality would have “have achieved [their] goal”. Inquiring the confectionery, their name stems from the composer Puccini itself because the Opera nearby was finished at around the same time while “it also sounded nice, Puccini Bomboni” (Brinkman 2024). Overthinking names to be meaningful is not a dutch virtue. However, Dutch virtue can be found in its imposing temporality dominating the life of not only its human citizens but also its tree citizens. Shortening both ‘usage’ and lifespan of trees hence controls the engineered Dutch landscape not only in space but also in temporality.
Fig Tree #
This Fig Tree was found growing under a bridge in one of the most touristic spots in the canals of Amsterdam. Due to previous climate conditions figs are not native in the Netherlands. Nevertheless once you become more aware, it is possible to spot fig trees every few hundred meters in the city of Amsterdam. Since the fig tree is one of the oldest of all cultivated trees, their residence in Amsterdam never occurs without their directly reciprocal relationship with humans. Besides a few exceptions, inhabitants of Amsterdam plant and maintain them in their private green spots maybe to get a hint of the Mediterranean in the local climate conditions. Yet, none of these trees are able to grow fruits that are ripe enough to convince birds into collaboration to spread their seeds and their genetics through reproduction. Fig trees found another way to generate offspring, again with the collaboration with their humans. This time through the logistics of globalisation and the appetite for fresh and non-local fruits globally demanding availability, anywhere at any season. A pedestrian next to the canals must have dropped the peel of an eaten fig on the floor, letting a left over seed flush into a gap of the bridge’s bricks and gladly finding soil that provided enough hold and nutrition to grow into a little tree. This is only possible since a few years, due to global warming caused by the human made climate crisis. Now this tree keeps growing and waiting for the climate to adapt for its needs to be able to produce ripe fruits to not only be dependent on its current collaborators, the humans.
Thanks #
Alphabetical order
Tree Support #
- Anna Tamm
- Lucas Evers
- María Mazzanti
- Lina Mittendorff
Sapling Support #
- Alexandros Sarantaenas
- Ana María López Gómez
- Arie de Fijter
- Arnoud Holleman
- Aurelian Ammon
- Bart Haensel
- Colin Schmid
- Davide Iozzo
- Emma Reeskamp
- Julian Schubert
- Kas Houthuijs
- Ksenia Gorokhova
- Ludwig Engel
- Maarten Smith
- Marcial Koch
- Rouzbeh Akhbari
- Špela Petrič
Seed Support #
- Barbara Visser
- Kadri Lind
- Mari-Liis Rebane
- Marie Valgre
- Moosje M. Goosen
- Mu Koch
- Pavle Mijuca
- Rasha Dakkak
- Taavi Suisalu
- Timo Toots
Organisations #
- Maajaam / Wild Bits, Vana-Otepää, Estonia
- Studio for Immediate Spaces, Amsterdam, Netherlands
- Waag Futurelab, Amsterdam, Netherlands
Bibliography #
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Bridle, James. 2022. Ways of Being: Beyond Human Intelligence. London: Allen Lane an imprint of Penguin Books. Brinkman, Ivor. 2024. “Where Is the Puccini Name Coming From?,” February 6, 2024.
Futurelab, Waag. n.d. “Waag Futurelab.” InteractiveResource. Waag Futurelab. Waag Futurelab. Accessed February 29, 2024. https://waag.org/en/open-wetlab-faq.
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