Tag Archives: Plant Growth Regulators

Plant Growth Regulators (or Phytohormones)

You’ve probably heard of rooting hormone powder, or auxin, or gibberellins – these are all ‘Plant Growth Regulators’. Plant Growth Regulators used to be known as ‘phytohormones’ which means plant hormones. This has been quite a contentious topic among plant biologists.

A hormone in an animal is a chemical messenger, a substance which acts as a signalling or control molecule to cause an action to take place. “Hormones carry out their functions by evoking responses from specific organs or tissues that are adapted to react to minute quantities of them”^ref. In animals, hormones are produced at a specific site (a gland, like the pancreas), work at low concentrations, and have a predictable dose-response. That is, an increase in hormone will result in the more of the related action (eg. more insulin leads to more sugar being taken up by the liver).

There *are* substances synthesised by plants which are involved in regulating growth – plant growth regulators – but they don’t work in the same way as animal hormones. It’s said that the assumption that they did sidetracked plant researchers for decades.^ref Plant ‘hormones’ are synthesised in multiple sites in a plant (and potentially in every cell^ref), have multiple actions on different cells (they don’t act on just one organ or tissue), don’t exhibit predictable dose-response behaviour like animal hormones and are involved in significant interactions or feedback loops with each other.^ref

What this means is that it’s quite hard to unpick what they do and how they work. The roles and mechanisms of plant growth regulators are still very much current research topics, as can be seen at two of the research groups at Cambridge University’s Sainsbury lab^ref1,^ref2 Some of the early theories about them were comprehensively demolished in a seminal article by Anthony Trewavas^ref (in particular the theory of auxin-derived apical dominance which was later proven wrong as explained below).

We now know that plant growth regulators act in concert with genes and the proteins they express, and not as an independently acting substance (one of the genes involved in cytokinin synthesis is known as LONELY GUY…).

So what are the plant growth regulators, where and how are they made? There are nine main plant growth regulators you may come across in your reading:

Auxin^ref – classically called ‘the growth hormone’ and a signal for division, expansion, and differentiation throughout the plant life cycle – involved in root formation, branching, the tropic responses, fruit development, shoot meristem function, the development of cotyledons and senescence. The most common form is Indole-3-acetic acid (IAA). Auxin acts in a ‘ying-yang’ relationship with cytokinin (see below)^ref as well as with gibberellins. More about auxin below.
Cytokinins (CK)^ref1 ^ref2 – involved in cell division, shoot initiation and growth (including maintaining the stem cell niche), nutritional signaling, root proliferation, phyllotaxis, vascular bundles, leaf senescence, branching and nodulation, seed germination, nutrient uptake, and biotic and abiotic stress responses. 6-BAP or 6-Benzylaminopurine is a synthetic cytokinin which is used in micropropagation and agriculture. Coconut water (not milk) has been found to be a natural source of cytokinins.^ref
Brassinosteroids^ref – involved in a wide spectrum of physiological effects, including promotion of cell elongation and division, enhancement of tracheary element differentiation, retardation of abscission, enhancement of gravitropic-induced bending, promotion of ethylene biosynthesis, and enhancement of stress resistance.
Gibberellins (GA)^ref – involved in multiple processes including seed germination, stem elongation, leaf expansion and flower and fruit development.
Strigolactones (SL)^ref – induce hyphal branching of arbuscular mycorrhizal fungi and are shoot branching inhibitors.
Abscisic Acid (ABA)^ref – involved in the induction and maintenance of seed dormancy, stomatal closure, and response to biotic and abiotic stresses.
Jasmonates (JA)^ref – shown to be inhibitors of growth but also involved in development of flowers and defense responses against herbivores and fungal pathogens
Salicylic Acid (SA)^ref – associated with disease resistance
Ethylene^ref – multiple effects on plant development including leaf and flower senescence, fruit ripening, leaf abscission, and root hair growth.

Slightly maddeningly none of these substances do just one thing – they’re all involved throughout the plant!

So how and where they are made in a plant? This isn’t simple either. In fact local biosynthesis is thought to be critical for plants, whereby plant growth regulators are made at the site where they are needed. For example both auxin and cytokinin are synthesised in leaves *and* in roots^ref, and can be made by chloroplasts and mitochondria, organelles which occur throughout the plant.^ref Chloroplasts can make precursors to auxin, abscisic acid, jasmonates and salicylic acid.^ref

Even though plant growth regulators don’t act in a predictable dose-response way like animal hormones, they still have a role in shaping plant growth in tissues which are sensitised to respond to them. Theoretically by understanding these responses we can manipulate a tree’s growth. And this is what they do in plant tissue culture (more on that below).

You may have heard the theory of auxin-controlled ‘apical dominance’, which holds that auxin produced by leaves at the apex inhibits lateral buds. This theory was strongly criticised by Trewavas in 1981: “The only hypothesis of apical dominance which has retained some measure of support is the nutritional one. A number of plants placed under conditions of reduced nutritional status adopt a growth pattern of strict apical dominance.” His point of view was further supported by a 2014 study which found that “sugar demand, not auxin, is the initial regulator of apical dominance”.^ref The researchers found that after removal of the shoot tip, sugars were rapidly redistributed over large distances and accumulated in axillary buds within a timeframe that correlated with those buds releasing. But auxin didn’t travel fast enough to be responsible for bud release. So basically they found that apical dominance arises because the main shoot is greedy for sugars, and due to its position at the end of the vascular system it can prevent lateral buds from taking the sugars needed to release and grow.

Auxin does play some role though, and the theory is that its role related to the fact that it’s the only plant growth regulator which displays polar transport. That is – it moves from the apex to the base of the plant, via the phloem, and can travel the entire length of the plant, ending up in the roots. This gives auxin a special role related to the spatial aspects of growth, and auxin ‘maxima’ (locations where auxin accumulates) are sites where new buds, flowers or lateral roots emerge. In fact, auxin and cytokinin work in concert throughout the plant, from shoots to roots, with apparently opposite effects in each location “like yin and yang”.^ref

An excellent reference point for this subject is the world of plant tissue culturing. This is where small pieces of plant tissue are sterilised and cultured in a medium containing plant growth regulators, which cause the tissue to grow into a ‘plantlet’ (sometimes in a test tube, if the source material is small). Further steps multiply the plantlet into several plantlets, which are then encouraged to create roots, multiplied again and/or planted out as seedlings to harden off. This process is used for industrial plant cloning where large numbers are required, and in the aquarium trade to avoid contamination with snails and other microbes (see Tropica’s website).

In plant tissue culturing, plant growth regulators are used to induce the relevant growth stage, which ones work for each species in which stage is documented in the ‘protocol’ for that species. In all cases specific ratios of cytokinin:auxin (and sometimes gibberellins) lead to different developmental stages – shoot growth, lateral shoot growth and root growth.^ref1,^ref2 To give you a bonsai-oriented example, one study determined a protocol for the micropropagation of Prunus Mume^ref. They were able to multiply fresh prunus mume shoots in a petri dish using a 4:1 ratio of cytokinins to auxins, and then root them using auxin.

So – apologies for the rather long read, it is quite a complicated subject! What can we take from all this for our bonsai practice? Firstly we can stop the brain-bending trying to understand how auxin controls apical dominance because it doesn’t – access to sugars does this instead.

Also we can use the yin-yang rule – high cytokinin:auxin encourages buds & shoots, and high auxin:cytokinin encourages roots. So I’m going to start adding some auxin rooting gel into my air layers and cuttings. Cuttings have never worked for me in the past so maybe this will be the secret sauce I need. I’m also going to try some cytokinin gel to encourage lateral budding on my trees.

If you are looking for products to give this a try, make sure the product actually contains the plant growth regulator you want. For example, Clonex contain auxin, and some of the orchid budding pastes such as Keiki paste contain Kinetin (a cytokinin). Many other ‘rooting hormones’ or plant hormones products on the market have no ingredient list at all, so avoid those. You can also find these products online in shops dedicated to hydroponics, where cloning and plant tissue culturing is a technique used by practitioners, or in lab supply shops such as microscience or Phillip Harris in the UK. You can even make your own hormone gels following these instructions.

Another trick you can use is that gibberellic acid can be used to break dormancy in seeds, if you really don’t have the patience to wait for natural dormancy to break. Or give coconut water a try, this has been found to have a similar effect in a range of species. For hard coated seeds in particular, usually it’s best to search Google Scholar for a researcher who has experimented with different approaches, since what works is very species-dependent.

Ramification of Branches and Foliage

After establishing trunk and branch structure, ramification (a fancy word for ‘branching’) of branches and foliage (as well as roots) is a key goal of bonsai. This makes a tree look older and more sophisticated, and gives the bonsai enthusiast options for continued development of the tree.

Ramification is created by branching the stems. Stem branching usually* requires buds, as a new bud creates a new stem. The pattern of stem branching for a particular species will depend on its ‘phyllotaxy’ (leaf morphology) and pattern of buds.

Usually in bonsai we don’t want more than two stems from the same location, the general guidance is to fork into two at any given junction. This is because strong growth of multiple branches at a junction leads to a bulging area on the trunk which bonsai judges don’t like. In the real world, many trees have reverse taper and bulging branch junctions though, so it’s your call. To avoid this situation, remove buds which are in places you don’t want by rubbing or cutting them off.

To improve ramification, you need to encourage as much budding as quickly as possible, then select the buds you want to develop. Pruning the growing tip is the main way to encourage budding, because pruning removes the apical bud (the dominant bud at the end of the stem), diverts resources into buds lower down the stem and sensitises those buds to respond to auxins and develop into shoots. In deciduous trees this should result in at least two buds generating from the stem instead of the one which was there. Another great way to create ramification on deciduous trees is through bud pinching – see Harry Harrington’s detailed explanation of how to do this. Bud pinching removes the entire primary meristem except for two outer leaves, this encourages the buds at those leaf axils to grow, along with two new buds at their bases.

Different species have differing abilities to respond to pruning, so try to get a sense by observing your tree of how well it will cope. Deciduous trees are designed for regeneration so in general they take pruning reasonably well, although if you take it too far they might send out suckers instead of new buds from the branches. With evergreen conifers you want to ensure there is some foliage and at least some buds remaining after you prune, otherwise it may not regenerate (unless it’s a thuja, or a yew, these guys are refoliating machines). I have cedrus seedlings in my collection and by cutting back the apical leader from not long after they germinated, and every year since, they have become extremely bushy and well-ramified (although, at the cost of developing a think trunk).

Gratuitous image of one of my cedar bonsai

Anything which stops or prevents tip extension will drive bud activation and ramification further back on the tree. In the case of conifers, the presence of flowers on the growth tips (as you see in juniper) has this effect as well, and can cause back budding. Lammas growth (a second flush in summer) can give you another round of ramification as long as you’ve pruned beforehand (otherwise it will just add to the existing stems).

Research has found that bud outgrowth is “controlled by plant hormones, including auxin, strigolactones, and cytokinins (CKs); nutrients (sugars, nitrogen, phosphates) and external cues”.^ref In particular the sugar sucrose has been identified as a key driver for promoting bud outgrowth and accumulating cytokinins – this is generated by photosynthesis.

In one study on apple trees, foliar application of a synthetically produced cytokinin 6-benzylaminopurine (BA) was found to generate three times the lateral bud growth on currently growing shoots compared to controls (but not on old growth)^ref and at the same time reduced the length of the main stem. BA was used to encourage better growth of bean sprouts in China before being banned^ref and has been shown to increase the number of leaves (ramification!) on melaleuca alternifolia trees^ref (melaleuca is the source of tea tree oil), and on some conifers^ref. Could BA (also known as 6 BAP) be useful in bonsai? You can (like most things) buy this product in foil bags on ebay, but there is a product in the orchid world called Keiki paste which also contains 6 BAP – so maybe some judicious use of ‘crazy keiki cloning paste‘ might also help ramification and shoot development in your trees?^ref You can also purchase BAP (as its also known) from vendors involved in hydroponics and suchlike as it’s used in in-vitro plant micropropagation.

If you baulk at paying £18 for 7ml of keiki paste, there is one other source of cytokinins which is a lot cheaper, more sustainable and clearer in its provenance – compost. This study found that compost created particularly from waste collected throughout spring^ref contained 6 BAP. Frustratingly there weren’t any free to read articles analysing compost leachate for cytokinin content, but if it’s in solid compost it’s a fair assumption there are cytokinins in leachate as well. Which makes me feel a lot better about the £300 I recently spent on a Hotbin composter! Which incidentally, produces gallons of leachate, which can be diluted and added as a liquid fertiliser.

* I’ve recently read a study which states that “apical meristems can be surgically divided into at least six parts and these then become autonomous apical meristems.”^ref What this suggests is that you could slice growing tips into 6 (or better, two since we don’t want more than two stems from a node) and they would become two stems instead of one! One to try next spring.

** By the way – it’s not auxins which cause apical dominance! Check out page 215 of this book, it’s nutritional status and phyllotaxy which determine the apical stem’s sensitivity to auxin which is present.

Buds

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Buds are the “small lateral or terminal protuberance on the stem of a vascular plant that may develop into a flower, leaf, or shoot.”^ref Buds are responsible for primary growth, and are created by meristem tissue (a meristem is an area of stem cells which differentiates into different types of cells).

If you look inside a developing bud, you can see the starting points of the different cells which will arise – they can be vegetative buds (shoots & leaves) or reproductive buds (flowers in angiosperms or strobilus/cones in gymnosperms). Below is an image of a Jack Pine terminal bud which has many lateral vegetative buds on the sides.

https://botweb.uwsp.edu/anatomy/images/budanatomy/pages_c/anat0999new.htm

When shaping your bonsai, you want to know where buds may appear, so that you can encourage the direction of growth and shape you desire. Predicting bud location is relatively easy in angiosperms, which follow a relatively reliable pattern in their growth. Bud growth is more unpredictable in gymnosperms, but many of the following guiding principles remain.

Firstly, there are different bud positions:

The terminal bud is at the end of a stem or branch and this is the growing tip which makes the plant grow larger.
Axillary buds develop along the stem during the annual growing season according to the architecture of the tree (see below for more); within this, preventitious buds are axillary buds which are dormant and then develop in a later season.
Adventitious or epicormic buds are buds which do not develop according to the repeating architectural pattern – they arise spontaneously from previously non-meristematic (growing) tissue which can be anywhere on the tree. They are unpredictable as described in this post.

Below are some examples of angiosperm buds. The terminal bud is on the end of the shoot, this comes from the shoot apical meristem (SAM). Then there are axillary/lateral buds which occur along the shoot – in angiosperms these develop in the leaf axils (a position adjacent to where the leaf is attached).

Bud behaviour depends on a tree’s architecture, which is genetically determined – that is, it will be very similar for trees of the same species, albeit also affected by the environment. There is a lot of research out there about tree architectures, much of it pioneered by Halle & Olderman in the 1970s, there is even a mathematical model which can be used to represent the architecture of a given species^ref. As explained in this excellent article^ref, “regular development of each plant represents the growth of repeating units – ‘phytomers’…a typical phytomer consists of a node, a subtending internode, a leaf developing at the node sites and an axillary bud (also called lateral buds) located at the base of the leaf”.

Each type represents a pattern consisting of a shoot with one or more leaves in the same arrangement. In some trees growth is repeated in a sustained way throughout the growing season (a single flush of leaves), whilst conditions are right. In others there are alternating growing and resting stages (multiple flushes of leaves). During the resting stage, new leaves and shoots are being created inside the bud^ref. I’ve copied some of the main architectural models into this post: Tree Architectural Models

An important part of the phytomer pattern is the leaf arrangement, known as the phyllotaxis. Leaves can grow singly at one position on a stem, or they can grow in whorls where two or more leaves appear at the same position arrange around the stem. When leaves grow singly they spiral around the shoot to optimise their light capture – apparently using the ‘golden angle’ of 137.5^o ^ref.

The leaf arrangement on your tree is important because each leaf axil (the base of the leaf) should be the location of an axillary bud (although in gymnosperms these can be missing). These are key to bonsai because they become new shoots (with leaves or flowers). They develop in the position just above where a leaf used to be; when it falls off, a scar is left and a bud generates above the scar.^ref In fact what is happening is a continuous bud genesis, so when you have a bud about to burst, it already has embryonic buds developing at its base – this is why buds look like they form at the leaf axil (in fact they formed on the previous bud). Your new branches and leaves will generate from these positions, and dormant buds may be located here. Read more about buds in angiosperms here, and in gymnosperms here.

The growth of an axillary bud (and its embryonic buds) can be suppressed by its neighbours – this is how ‘apical dominance’ works. It used to be thought that in apical dominance, the shoot closest to the sun emitted hormones which suppress the growth of buds lower down the plant, ensuring that it gets the most resources. This research group at Cambridge University study the development of axillary shoots and their research says “shoot apical meristems compete for common auxin transport paths to the root. High auxin in the main stem, exported from already active meristems, prevents the activation of further meristems”^ref. This results in axillary buds going dormant and becoming ‘preventitious’ buds, but they are still available to grow later if conditions change. According to this article, apical dominance in trees only works on buds in the current year of growth due to the slow movement of the hormone auxin through the tree^ref – meaning that current year buds on a branch are suppressed by the terminal bud on that branch and not by the main leader. HOWEVER, it has recently been found that auxin does not move fast enough to have this effect, and instead it is driven by sugar flows to the apical meristem.^ref The effect of apical dominance remains, however it is now thought that sugar flow drives this and not auxin directly.

Encouraging axillary bud growth is a way of increasing ramification on a bonsai, as it can create multiple shoots instead of just the terminal buds. If the terminal buds are removed, axillary buds get the chance to grow, often more than one.

Application of exogenous cytokinins (benzyladenine) has also been shown to increase bud initiation^ref (see my post on ramification of Branches and Foliage for some substances containing benzyladenine).

Equally, looking at how the leaves are arranged, you can work out where new shoots will arise from existing stems. By removing the buds or shoots not meeting your design, you can encourage shoots to grow in the direction and position that you want. But it’s not enough to know about bud position, you also need to know what kind of bud is present – a vegetative or a reproductive bud, and you need to know the difference between short and long shoots – more here.