Advanced Propagation Techniques (Root & Shoot Setting & Hormone Treatment)

Here I shall share my findings from the last 5+ years of working in a intensive production environment producing cuttings & growing vegetative material. I am going to detail HASAP methods essential to keeping a perpetual mother library running & consistently keeping stock of propagules & the methods & processes involved in maintaining consistency in cultivars.

I shall provide a detailed process for cleaning, preparing & grading propagation materials for the various applications. I shall share my own sterile propagation medium, a recipe/formula plus a step by step guide for inoculating new propagation material, applying (RSH) "Root Setting Hormones" for keeping a supply of organic cannabis through a mixture of various techniques & applied cultivation processes that I have learned over the many years & found to be optimum when put into in practice.

Well it has been 2 minutes. Where is it?

I have to type it up & do an edit on word & I will keep taking step by step photos & keep a propagation diary to illustrate the importance of these processes by allowing pythium & other vectors to manifest in control samples for educational purposes moving onto more advanced stages as the season develops.

For now what we will start with is the basics in selecting suitable propagation material & the fundamental principles to good propagation propagation practice.

Remembering the 7 P's..

I know I was just ribbing you.

The first step to creating pristine cuttings is growing a mother plant or (DP) "Donor Plant" under optimum conditions free from P&D "Pest & Disease" These conditions vary depending on morphology & genotype & the season but is considered anytime shoot elongation is not taking place.

Essentially we are looking for clean material with suitable pliability/plasticity with rapid dividing mitotic cells.

Seed plants are a good place to start but remember that small insects can live within the seed coat & this causes them to rapidly loose all vigour, most of the time small biota is the cause of such viability issues & can later manifest themselves in the juvenile plant, therefore these seed plants are not suitable as (DP) to supply cutting material until cleaned up.

However quick grown resistant plants performing mitosis are able to out grow pathogens before they can even become vectors.

First of all let me just say cleaning cuttings is only something you really need perform if you are failing initial laboratory tests, here tests are cheaper to confirm presence of issues before spending money time & energy cleaning something that has been in transit for long periods & showing a mere lack of luster. Some of these expensive tests will fail due to the presence of microbes from organic soil or the presence of moulds from transport, even too much yeast when testing curing flower biomass fails the tests, however for this purpose we are only testing the vegetative materials for the presence of pesticides & fungicides & other substances that may be harmful to health or prevent successful propagation.

Maybe you wish to regenerate a cutting that has been in stasis for a long time or mistreated or so you are led to believe.

Maybe it's just crappy.

Plant materials can be a-sexually propagated indefinitely without degradation providing they are looked after & quick grown, then using basic cloning methods is the optimal way to cultivate or multiply ex vivo.

The problem with advanced methods is the need for solvents chemicals & other substances that are not ideal in smoked or eaten crops. Cannabis is unique in that it has many ways to consume it that makes it open to more scrutiny than another plant we use.

The idea of having to apply a fungicide or a mix solution to get a jar or cured heads is insane to me..

With cleaning materials concentration and length of time for absorption are critical some of these substances can take generation to remove before you precious cultivar is suitable for the retail sector.

Some species will require only a brief treatment with low concentration whereas others may require a prolonged treatment with a more concentrated dilution.

Alcohol is toxic to the plants as is bleach, too long time in a concentrated solution containing either may kill the cuttings on the mechanical stirrer if you forget because you are stoned. Remember this is supposed to be clean, you need to not be smoking in the same areas as this propagation happens. Especially if you smoke tobacco as this can pass a virus onto your propagation material.

For rational application, dip a number of cuttings at the time in a mechanical bath, not one by one.

Ichabod Crane said:

Well it has been 2 minutes. Where is it?

Click to expand...

Don't you have a roof to do somewhere???
Let the guy alone and let him drop some knowledge.
Don't mind Ichabod he is used to people running round headless.

Please carry on this will be good.

Here we have a finished NL 'Big bud' cutting or the Blues as it is called in the uk.



Here is a tray of 'Nodal' NL BigBud cuttings in a Pre-charged puck. These pucks can be tailor made to suit your unique requirements & can be inoculated or ameliorated with multiple stimulants to enhance rooting.

Process; Cuttings are struck into a pre-made hole & heeled in to ensure that the stem is in contact with the culture medium & maintain it's turgidity in conjunction with the use of a dome to elevate the humidity allow some gaseous exchange & maintain stable environment for the first few days.

Warm tops & cool bottoms triggers the plants to 'callus' near where the cut/cuts was made & produce root structures.

These structures depend on the propagation material or media. Some structures are suited to various cultivation methods so it is wise to choose one that suits the methodology. Water roots for sucking up grape soda & organic feeder roots are an example of different structures & mixing them up will slow down a plant & change it's phenotype.

Some substrates come with a naturally occurring inoculant/rooting enhancer (RSH) "Trichoderma" in coco coir is an example of such.

Organic coir cuttings develop extensive fibrous root systems more advanced & than that of peat & (RW) "RockWool".

They are more tolerant tolerant of water saturation & they produce healthier cuts more ecologically than (RW).

Rockwool is great but it has no advantages in the organic system beyond microbial cleansing, it is non reusable or biodegradable & plants have to develop organic roots when transferred into organic dirt wasting (ATP) "Adenosine triphosphate" taking more tile to kick off & grow healthy plants.

(Choose a media suitable for your production to combat virus or pathogen)

There are 100001 ways to take cuttings remove the epidermal layer & expose the cambium we shall not go into those here @ this time.

Optimal temperatures show an increase in % success rate & keep Phytophthora @ bay, this is your main issue/enemy with basic method of cloning, using the right cloning material @ the right time & choosing the right type of cut can maximise your productivity.

I have done a thread on the types of cuts & propagation used applicable to cannabis.

I shall put a link to it here:

The 3 that are most relevant are "LeafBud" "Heal" & "Nodal" cuttings. All have distinct advantages for the material selected from the (DP)

I have pic's in my albums from the linked thread but I can update these & show how to dissect the tips of the plants & grow them but for now lets concentrate on LeafBud cuttings as they offer the best advantages IMHO for classical cloning or (TC) "True Clones"

To throughly clean plant material from the (DP) we must use a 4 stage process.

Step 1 is to clean the mother plant up to encourage pliable growth.

Remove any visible vectors, these can be seen with a loupe or viewing glass, mites, thrips, eggs, larva, soil particles or other foreign bodies should be avoided, weak growth & detritus discarded, the main wash or 'cleanse' of the (DP) is with fresh mineral water or DD Double Distilled Water followed by a dedicated UV light exposure in conjunction with blade treatment or a combination of all 3 if the material is contaminated & then "Quick Grown" to get suitable Explant material, otherwise we are going to use a 2 part solvent dilution depending on the level of contamination to bath the "Explants" & rinse them 2 times in DD or mineral water using a Mechanical Stirrer.



Next we need the 1st of our flask glassware the proper Heisenberg kit.

Erlenmeyer Flask, formally known as the BrE



It is handy to have the chemicals labeled in appropriate measurement dispensers such as "Wash Bottles"



It is wort mentioning here that the light can be used to sterilise all the kit prior to use. I would suggest 2 types of light one is handheld the other is a fixture for performing the "Autoclave" phase prior to starting & for cooking/sterilising propagation/culture media recipe.



Be sure to wear gloves without residue to handle the materials & use eyewear when handling these chemicals/solvents.

Everything needs to sit under that light for 45 minutes except the plants, that will kill them..

@ this stage you get the idea of how clean this needs to be how slack most of you are & you should have some idea of the risk factors the process involved in bacterially cleaning up plant material & the propagation kit.

Most the essentials we already have as indoor gardeners a propagation tent such as the CR70 or a 1m2 tent is suitable for 'Autoclave'.

You need to have a HEPA filter & a germicidal lamp





This cleaning should be used for all propagation cultures basic or complex even if reestablished into manure or similar substrate.
Cleaning & maintenance is fundamentally essential to plant vigour & keeping stringent conditions..

Next I shall talk about the combination of sterilisation fluids & how & why to apply them, the various (RSH) solutions for rooting in the flask, initiation & the recipe ingredients & the nutrient formula for the liquid culture medium & the hormone application process. We don't need to use Murashige and Skoog for cultures, sugars feed bacteria & if you don't clean explants properly certain culture medium won't help & it will mean you needing to buy additional WoooWWoo..

Thidiazuron for example has shown to be ineffective in creating callus in 100% of cannabis tested.


Remember there is lots of Woo Woo juices in cannabis growing. Stoners will buy any fancy labeled antioxidant or wetting agent..

I am going to show you how to create a medium from scratch of known constitution & be able to mass produce this culture medium for billions of explants, establish callus in seedlings and maintain viability in all tests for a year in 52 passes.

The sterilisation liquids can vary in potency anything from D.D water (Double Distilled) or various elutants can be used from 20-1 to 2-1, further enhancements to the dilution like wetting agents that can be added to reduce bath time along with other treatments like 'Augmentin', which is basically Amoxicillin to boost to the explant material activating (SAR) before commencing to initiation.

The goal with the bath is too clean the material but not degrade the material you have spent time nurturing. There are some other additives like citric acid that can help prevent discolouration of explants if you are doing bulk or mixed workloads.

This is where the testing comes into it again. The ultimate goal of the wash is of find the optimum level of dilution of the least toxic nature & substances with the minimal costs to remove the contaminate, however to achieve this you have to have laboratory analysis of known contaminants.

Just because your cultures survive doent's mean you cleaned/scrubed them. Or that you even needed to without the analytics.

Common scrubbing agents are Clorox Bleach & Sodium Hypochlorite & Ethanol they are diluted to the requirements of the plant material/technician they are prepped & washed with the mechanical stirrer in a combination of D.D water & the wetting agent/detergent and stirred enough to release the Saponins before the liquid is discarded & the propagule explants are rinsed in either a lesser elution or rinsed or bathed again in a multi step wash of 'Augmentin' & Citric acid.

Some of the additives to the bath can have reactions & cause heat stress so a little knowledge of chemistry is needed as you can easily damage & poison your cultures or yourself.

Generally speaking leaves and embryos tolerate mild sterilant, roots & nodal explants need comparatively high strength sterilant, all material is different.

There are 3 stage & 4 stage washes that incorporate an ethanol alcohol disinfectant it is advised to use 70% ethanol, this reduces the risk of the explants succumbing to the degradative effects of either the Bleach & the Alcohol or a combination of the 2 & this step is only needed for cleaning the material not keeping explants in a library

The cultures are generally triple rinsed in Autoclaved mineral water or D.D water & then rested on filter paper in wait.

These 'washed' explants are then ready for initiation into a culture medium you shall prepare in an autoclaved glove bag, inside your sterilisation chamber.

Does anybody else feel like there should be inline images that aren't appearing?

I think your right Agent Pothead. I'm busy ATM I've got lots of gardens I'm getting prepped for the auto flowers. The camera has been in use I have not got it here to do the uploading.

I have to start from scratch with mums & cannot keep a big mum room so this is how I am going to deal with the matter.

I've not made the Nutrient Agar yet as I have nothing to initiate but I have been growing up seed plants & I am sure another delivery of snips will get processed ASAP.

Winter is not the time to propagate cuttings. I'm looking to see what the turn around time is after Brexit vs now. Currently only the UK can serve Europe as a clone mothership as the post is smooth and uninterrupted. You cannot say that about another European postal service or couriers.

The Hotplate stirrer I have I share & it is in use @ another property making MTC oil for a patient of mine & It will be busy for a few weeks till I can use the same one for cloning. I did find one without the heat for £30 on Amazon today tho.

I've not got a light up yet, I just received a box of bits from the UK. The 'essentials' what would you take to a desert Island? It must fit into a shoebox What do you take?

I am stocking the Propagation house with the 'essentials' so that the next few years runs smoothly..

Here is a new & soon to be released culture flask kits specifically designed for the cannabis industry equipped with lights, they're stackable modular systems & can run battery operated so shipping is gonna be possible with this.

March they're on release to the general public, get some & have a fiddle.




Big Box Modular Growth System & the GA-7 Competing Vessel

I am going to make my propagation media inside the vessels. They both support Liquid & Gel Medias




Modular Big Box System.

Custom or bespoke vessels available, three times as tall. Ports for gas injection or chromatography. An opaque bottom. Side LED lighting. Whatever your need to work they will help you to design a custom solution.

They also sell a Rocker to go with a modular kit so with a liquid media you can immerse cuttings on a time schedule a bit like ebb & flow



This is the root stand you suspend explants for immersion into the media.



Micro-Rocker

You make these Mate Dave . Released to the public where from any one else tried them yet please excuse the question but they look ok look handy for sure.

I don't make them no Darksider, I am not on the product development team either but I have been giving feedback to them from basic product testing offering advice & troubleshooting a few options that may become wholesale in the future.

If I want to start a business using them what price can I get them for.

Can I have a set designed specific to my needs that I can stock for resale.

I seen these kits in the lab I have been watching for a year or so. They are good practical no spill Vessels. The micro prop company didn't want to pass the name of the manufacturer on or their procedures for cleaning & initiation but I did more research, asked about how & why they use different media & spoke about my work experience with micropropagation & what I have done for the last 15.

Long & the short of it is I'm cute, I learn fast and it's hard to pull a fast one on me & I even learn the stuff that they didn't mean to speak about so bit I have built up/refined the list of equipment needed from a bag of junk to this multipurpose laboratory setup that is as practical & adaptable in the field as it is the lab or shipping plant material..

A no nonsense approach to cannabis cultivation.

So it's probably worthwhile talking about the various suspension medias that are used for micropropagation & more specifically cannabis/hemp micropropagation.

Each have their benefits and drawbacks. Removing cultures from gels can tear roots & residuals can cause bacterial contamination when passing or transplanting.

There are other suitable suspension media like the tray listed above, sterilised micro glass beads & coconut coir rice are sometimes used.

Nutrient Agar commonly known as "Murashige & Skoog" - Most suitable to holding cultures in flasks in a dormant phase or for growing shoots as the liquid nutrients are less bioavailable in these types of medias helping to control or suppress growth. Liquid culture media provides better oxygenation for root formations & rapid growth & they will rinse & transplant trouble free however they require some sort of support

It is common for explant cultures in a gel media to have a liquid nutrient added when cultures begin to acquire a need for supplementation.

Of course once your shoot propagules are rooted you can use the magnetic stirrer to rinse them of any residuals with a triple DD wash before transplanting into RW or Organic soil.

good stuff...Good to have some dormant stuff in agar.. Slow & steady while everything else is daily input requiring but most rewarding/efficient...

You can probably tell Dr it's good to perform cleaning every so often as you will find that cultures sat in stasis will not have the rapid cell growth to outcompete infection. @ no stage in the process of cleaning or removing virus or other vectors should ex vivo plants not be kept growing.

Micropropagation is a means of reducing propagation space & storing plants until they can be hardened off & transplanted..

Explant root formation can be promoted by pretreatment with root setting hormones (RSH) before they are inserted in the sterile suspension rooting medium.

The chosen compound is often mixed with DD water to a concentration of ≈ 20–200 ppm (parts per million). The explants are then bathed in the solution for upto 24 hours & then rinsed for 4-5 seconds in DD water.

The compounds can also be dissolved in a 50% 70-% alcohol concentration @≈ 500–10,000 ppm to save bath time exposure however as discussed above, benefits & drawbacks apply to these multiple washes.

Root setting hormones can be made into a homemade solution of the active compounds (Indolebutyric acid (IBA) or Naphthaleneacetic acid (NAA) For the specific application of these compounds reference should be made to the individual labels.

Certain precautionary procedures should be followed when using RSH.

Fungicides such as 'Captan' & antibiotics for particular treatments can be mixed with the RSH, it will save one work portion. Sucrose & growth retardants or other (RC) Rooting Cofactors can be applied. The gel media is enough to hinder excessive growth for storage.

Things to remember; RSH is best absorbed from a newly cut surface therefore if the explants have been kept or stored for some length of time, a new fresh cuttings should be made to the Basal/Proximal end.

Concentrations & exposure time for absorption are critical. Some explants will require only a brief treatment with low concentrations whereas others may require a prolonged treatments with a more concentrated dilutions.

If the Explants have been 'Scrubbed' with an alcohol bacterial wash & disinfected with Bleach & treated with & Erythromycin Amoxicillin Penicillin or similar then we can move onto the next phase & not have to have these antibiotics in the rooting media & they can be reserved for pretreatments & after treatments.

Liquid RSH Media

To make our solution for sterile Explant culture we first need to Autoclave some DD water. To the water we shall add;

1.5ml Per US gallon Canna Aqua Vega A & Flores
1.5ml Per US gallon Canna Aqua Vega B & Flores
3 mL Superthrive per 10 gallons
0.5 mL Clorox Bleach per 10 gallons

These should be thoroughly mixed in the stirrer & heated to 15-18c so that the solution doesn't cause any nastic responses from the explants.

Make sure to pH the solution to 6.0 whilst mixing all the above products on the stirrer.

It is important to note that the inclusion of the Flores part of the nutrient actually encourages root setting while the Vega actually suppress's wound root initiation or differentiation, preventing callus formation.

Dedifferentiation
The capability of previously developed, differentiated cells to initiate cell divisions to form a new meristematic growing point.

Wound-induced roots

Wound-induced roots develop only after the cutting is made, in response to wounding in preparing the cuttings.
These roots are formed de novo (anew)

ROOT INITIATION

There are 4 steps to Initiation
1. Dedifferentiation of specific differentiated cells

2. Formation of root initials

3. Development of root primordia

ROOT GROWTH

4. Growth & emergence of root primordia

Auxins

Auxins – natural & synthetic
Indole-3-acetic acid (Natural)
Considerable auxin activity, easily metabolised by the plant.

Indole-3-butyric acid (natural)
More effective than IAA for rooting, not easily metabolised by the plant.

1-Naphthaleneacetic acid NAA (Synthetic)
More effective than IAA or other's in the auxin family for rooting. However registered as a pesticide, although commonly used in tissue culture it can be detected by HPLC-tandem mass spectrometry.

Auxin may or may not be required for initiation of roots on stems, it has been shown that divisions of the first root initial cells are dependent upon either applied or endogenous auxin, showing us genes responsible for making plant tissue respond to auxin are more critical than those producing auxin.