Research The nexian phalaris breeding programme

[Grasshoppers]

Practical Approach to TLC Analysis of Phalaris Alkaloids

This guide outlines the Grasshopper TLC method for analyzing the alkaloid profile of plant materials, specifically Phalaris species. The procedure allows for quantitative analysis of small samples without requiring costly equipment, distinguishing it from standard qualitative TLC approaches. Its high sensitivity relies on the natural fluorescence of DMT, eliminating the need for colorimetric reagents. Though optimized for Phalaris, the method is adaptable to other samples.

Sample Collection and Preparation

• Collection: Harvest around 500 mg of plant material (e.g., mixed leaf segments) to account for uneven alkaloid distribution.
  
  
  
• Drying: Prefer microwave drying for efficiency.
  
  
  
• Weighing: Use a dry weight of 50 mg for processing.
  

Extraction Process

• Initial Boiling: Boil the sample in 1% acetic acid for 15 minutes, then soak for at least 60 minutes in the hot solution.
  
  
  
• Dilution: Dilute the extract to a concentration of 1 mg plant material per 1 ml of 1% acetic acid, yielding 50 ml of acidic extract.
  
  
  
• Extraction Vials: In a 20 ml vial, mix 1 ml dichloromethane (DCM) with ~40 mg sodium carbonate (Na₂CO₃).
  
  
  
• Alkaloid Extraction: Add 100 µl of the acidic extract, shake vigorously to enhance alkaloid transfer.
  
  
  
• Phase Separation: Add another ~80 mg of sodium carbonate to the vial and shake again to break the emulsion and dry the DCM. The sodium carbonate does not fully dissolve in the water, adheres to the vial walls, attracting and binding water droplets. As a result, only the DCM remains as a liquid, while the water is absorbed by the undissolved sodium carbonate and held to the vial's walls.
  

TLC Spotting and Development

• Spotting equipment: Use glass syringes with fine needles (30 gauge) and cotton in the syringe neck to stop sodium carbonate particles.
  
  
  
• Spotting: Pour the DCM from the vials into syringes to apply it onto 10 cm x 5 cm silica 60A non-fluorescent TLC plates. To prevent needle clogging from alkaloid residues after DCM evaporation, attach glass capillary tubes to the syringe needles. Maintain the TLC plate at 45°C using a hotplate to aid evaporation. While DCM may spread during application, it does not elute DMT, ensuring the spots remain small and concentrated.
  
  
  
• Development: Place the TLC plate in a chamber with methanol (99.9%) and NH₄OH (25%) in a 39:1 ratio. Development takes about 20 minutes.
  
  
  
• Visualization: Expose the wet plate to 275 nm UVC light in a dark box, then take standardized photos with prolonged exposure of both wet and dry plates.
  

Analysis

• Fluorescence Detection: On wet plates and dry plates, DMT appears as fluorescent spots of different colors when exposed to UVC light at a wavelength of 275.
  
  
  
• Differentiation of DMT derivatives: The exact colors of the intrinsic fluorescence of the DMT derivatives under UVC light at a wavelength of 275 nm are shown below. Background light from the UV emitter has been filtered out to enhance visibility.
  
  
• Retention Factors (RF): The RF for the different DMT derivatives are:
  N,N-DMT: 0.50
  5-MeO-DMT: 0.48
  5-HO-DMT: 0.50
  unknown substance: 0.50

Additional Notes

• Reagent Considerations: Use sodium carbonate instead of sodium hydroxide to avoid damaging 5-MeO-DMT.
• Solvent Alternatives: DCM could be replaced with locally sold petrol ether, though maybe not all brands are suitable.
• Sensitivity: The method detects alkaloid spots as low as 10 ng to 1µg, with accurate quantification across this range.
• Other compounds: UVA light at a wavelength of 365 nm can be used to stimulate fluorescence of other compounds such as betacarbolines.
• Densitometry: Detailed guidance on plate interpretation and alkaloid quantification via densitometry will be addressed in a future update.

For any further details or updates to the methodology, please reach out. This protocol will be revised as needed based on ongoing research developments.

 

[neurobloom]

I have just acquired 12 Tunisian wild accessions of phalaris aquatica from 12 distinct regions with varying soil types, yearly average precipitation and climate. There are 50 seeds for each accession. That's a total of 600 seedlings to undergo phenotypic selection for high alkaloids and clean profile.

Now the question is:
- what's the likelihood of finding a super potent clean 5-meo-dmt and DMT clones ~1%+ out of these 600 seedlings?
- what's the yeild ceiling that can be achieved through selective breeding of the two selected highest yielding seedlings from the 12 accessions?
- how far said ceiling can be raised by introducing the top yielding selected foreign genetic material from grasshoppers collection?

To give more perspective, the Tunisian phalaris aquatica cultivar i tested yielded between 0.3% and 0.4% of relatively clean 5-meo-dmt by dry weight after a cleaning step on the crude freebase. That's the average for a cultivar that was confirmed to have been bred for low alkaloids. imagine what yield can be achieved through selective breeding from within the same breeding population that was used to create this low alkaloid cultivar.

Let that sink in for a moment...

 

[Grasshoppers]

A Critical Setback for the Project :

The project has encountered a severe setback. Previously selected high-yielding plants are now dying, and the potential loss could be immense. Efforts are being made to rescue the plants, but the best approach remains unclear.

Events Leading to the Problem:

To protect the most valuable plants from the cold season, they were moved indoors by different breeders at various locations. However, after spending some time indoors, these plants have started to rot.



Strangely, the dying plants are located right next to healthy, freshly planted seedlings growing under identical indoor conditions. Meanwhile, clones of the same plants left outdoors in the cold are thriving—although not actively growing, they remain healthy and green.

Insights and Conclusion:

This phenomenon highlights the powerful circannual rhythm in Phalaris aquatica. The species appears highly sensitive to disruptions in its seasonal cycle. This incident underscores a critical lesson: Never move Phalaris aquatica indoors during winter.

 

[aizoaceous]

I'm sad to see this, and hope you don't lose those genetics. If you do, then I hope it's at least easier to re-collect them from nature, guided by what you've learned so far.

Almost nothing seems to be published on the culture of Phalaris in a controlled environment, since nobody needs that except the handful of commercial or academic groups breeding pasture grass (and even they seem to mostly use greenhouses not too different from natural conditions). My P. brachystachys has grown and set seed in my artificial conditions, but my P. aquatica died before setting seed on two attempts. I'd thought that was because it was root-bound, but it might have been the absence of temperature, photoperiod, or other seasonal cues.

Curiously, both @Grasshoppers and I have failed to grow Phalaris in coir hand-watered with hydroponic nutrient solution. I've had success with the P. brachystachys on a flood table, operated either daily or every two days, hand-watered every week or two in order to flush out salts. I used pots of 50/50 coir/perlite about 3 inches deep, EC ~ 2.0 mS/cm, pH ~ 5.8. Photoperiod was 16 hours, under white LEDs around 15 klux. I don't know why the hand-watering failed, since most plants would be fine with that. It's not excessive dryback, since my hand-watered pot was deeper (about 9 inches) and actually dried back less than the shallower pots on the flood table. I'd thought it might be insufficient dryback, but extending the hand-watering interval looked worse if anything. Weak plants moved from the hand-watered pot to the flood table became stronger.

My hydroponic conditions were accidentally very good for alkaloid production, consistently yielding around 1000 ug/g fresh weight of 5-MeO-DMT, perhaps since they're still somehow stressful. The same seed planted outdoors is growing very well, with wider, stiffer, and longer leaves but yield around 200 ug/g. It's possible that low soil fertility explains that, so I'm now injecting fertilizer into the irrigation water and will retest soon. I'll also look for improved extraction methods for the weaker grass, since my freezer is quickly filling up.

 

[Grasshoppers]

I'm sad to see this, and hope you don't lose those genetics. If you do, then I hope it's at least easier to re-collect them from nature, guided by what you've learned so far.

The first plants moved outdoors are showing signs of recovery, though a loss of genetic material still seems likely. While we have retained a significant amount of genetic material, the efforts invested in cultivation and testing could be lost.

Almost nothing seems to be published on the culture of Phalaris in a controlled environment, since nobody needs that except the handful of commercial or academic groups breeding pasture grass (and even they seem to mostly use greenhouses not too different from natural conditions). My P. brachystachys has grown and set seed in my artificial conditions, but my P. aquatica died before setting seed on two attempts. I'd thought that was because it was root-bound, but it might have been the absence of temperature, photoperiod, or other seasonal cues.

Considering the recent insights into the seasonal rhythms of Phalaris, this explanation seems very plausible.

Curiously, both @Grasshoppers and I have failed to grow Phalaris in coir hand-watered with hydroponic nutrient solution. I've had success with the P. brachystachys on a flood table, operated either daily or every two days, hand-watered every week or two in order to flush out salts. I used pots of 50/50 coir/perlite about 3 inches deep, EC ~ 2.0 mS/cm, pH ~ 5.8. Photoperiod was 16 hours, under white LEDs around 15 klux. I don't know why the hand-watering failed, since most plants would be fine with that. It's not excessive dryback, since my hand-watered pot was deeper (about 9 inches) and actually dried back less than the shallower pots on the flood table. I'd thought it might be insufficient dryback, but extending the hand-watering interval looked worse if anything. Weak plants moved from the hand-watered pot to the flood table became stronger.

For hydroponic cultivation, P. arundinacea might be a promising candidate. It can thrive in deep water culture and even tolerates anaerobic root conditions. We plan to conduct further screenings of P. arundinacea specimens to explore its potential. So war we only found one promising plant of this species.

My hydroponic conditions were accidentally very good for alkaloid production, consistently yielding around 1000 ug/g fresh weight of 5-MeO-DMT, perhaps since they're still somehow stressful. The same seed planted outdoors is growing very well, with wider, stiffer, and longer leaves but yield around 200 ug/g. It's possible that low soil fertility explains that, so I'm now injecting fertilizer into the irrigation water and will retest soon. I'll also look for improved extraction methods for the weaker grass, since my freezer is quickly filling up.

How does the concentration of other alkaloids compare? Have their proportions shifted as dramatically as the total yield? We’ve observed that fertilization can enhance alkaloid content, though we haven’t verified this experimentally. Testing the effect of fertilizer on alkaloid levels sounds like a valuable experiment.

Boiling leaves in dilute acetic acid, followed by basification and extraction with non-water-miscible solvents, works effectively. However, the formation of emulsions can be frustrating. If you find a more convenient extraction method, we’d be very interested in learning about it.

 

[aizoaceous]

How does the concentration of other alkaloids compare? Have their proportions shifted as dramatically as the total yield?

I've superimposed five chromatograms of P. brachystachys extract, normalized to the same 5-MeO-DMT peak height: that outdoor batch, an indoor batch (also from many individuals pooled together), and analytical samples from three randomly-chosen individual seedlings.



So the indoor samples agree very closely, while the outdoor sample shows relatively higher DMT and lower gramine. The difference doesn't seem practically important here, since the dose of DMT is negligible either way. This does seem to show clear environmental influence on the alkaloid ratios, though. That and my ~5x absolute yield may make indoor culture more attractive to me, though from your result here that may be impossible for P. aquatica.

 

[neurobloom]

That's really unfortunate about the clone losses. It's worth mentioning I never had any phalaris losses in semi arid Mediterranean climate.
These are 27 days old seedlings of wild accessions. Looking pretty healthy and strong.

Clones are also growing super fast in the cool of winter. Growing bigger even after three harvests so far. I haven't tried coco coir but they grow pretty well in peat moss.

Recently I've tried acid base on tanit strain pulling with boiling n-hexane . While it was pretty inefficient at pulling actives the extract felt like pure 5-meo-dmt. Hexane clouded up very white when reduced and crashed a faint amber clear oil. Vaporizing this freebase on a piece of foil didn't leave behind any carbon residue which suggests high purity.. dose in the range of 3mg was already pretty potent.

 

[neurobloom]

Forgot to mention that pulling with boiling hexane results in a freebase with purer bioassay qualities of 5-meo-dmt than with chloroform. Room temperature hexane didn't pull anything. Even when it's boiling hot it was only able to dissolve little freebase.

I evaporated the pull too fast and crashed an oil but i could have gotten crystals if i cooled it slowly in a controlled way like a glass jar inside a thermostat or something. Pulling with chloroform subsequently still yeilds more freebase.

This confirms that hexane should only be used as last step cleaning mini acid base for crystalization and a wide spectrum solvent like DCM, chloroform, xylene or toluene should be used to pull initially from the crude tea to maximize yield.

 

[neurobloom]

This is the hexane pull crashing out 5-meo-dmt oil after rapid evaporation and cooling to room temperature. It clouded a lot initially before crashing a light amber potent oil

 

[Grasshoppers]

Improved TLC Process for Alkaloid Analysis

This season, we have streamlined our Thin-Layer Chromatography (TLC) process to enhance efficiency and accuracy in plant alkaloid research.

Sample Preparation:
25 mg of dried leaf material is soaked in 1 mL of methanol with 25% ammonia in a 39:1 ratio.
Minimum soaking time: 8 hours, in complete darkness to prevent photochemical degradation of alkaloids.

TLC Procedure:
Samples are manually spotted onto non-fluorescent, unmodified silica 60A TLC plates using a 25-gauge blunt syringe needle.
The plates are developed using the same solvent mixture as in the extraction step.

Fluorescence Imaging & Analysis:
UV fluorescence images are captured under 275 nm and 365 nm UV light, for both wet and dry plates.
Image processing is performed using a custom C++ script with OpenCV, designed for speed and precision.
- Removes baseline color, enhancing plate visibility.
- Extracts and analyzes sample regions.
- Performs densitometry to quantify alkaloid content.
- Saves processed images and exports CSV data with color density metrics.

This script will be freely available for those interested in advancing plant alkaloid research.

Compounds Identified & Quantified:
-N,N-DMT
-5-MeO-DMT
-5-HO-DMT
-NMT
-5-MeO-NMT




Efficiency Gains:
Processing time per sample: ~3 minutes
Cost per sample: ~$0.25
The increased efficiency will allow for a much higher sample throughput.

Research-Only Commitment:
If you're solely looking for a recreational experience, Phalaris is not yet the right path. We are committed to research and breeding, and seeds/clones are shared only with individuals demonstrating a genuine interest in furthering Phalaris studies.

Join Us:
If you’re interested in contributing to this effort, please contact me or @neurobloom.
Let’s push plant alkaloid research forward together!

 

[Jamie01]

Hey this is really great what you are doing here! It’s inspired me to start growing out individual aquatica and arundinaceae seedlings to test later. In the past I always seeded entire pots and just went with it. I think y’all are cracking the Phalaris code here.

 

[Grasshoppers]

Analysis has commenced. A total of 18 samples were processed today.

• Sample Material: 25 mg dried leaves
• Extraction Method: Soaked in 1 mL ethyl acetate (EtOAc) with concentrated aqueous ammonia (25%) for 8 hours
• TLC Plates Used: Macherey-Nagel ID 818161
• Eluent System: Methanol (MeOH) with concentrated aqueous ammonia

Visualization Conditions:

• 275 nm (wet)
• 275 nm (dry)

Plate 1:




Plate 2:




Conclusion:
aq_irq_02 is high in NMT
aq_irn_02 contains an unidientified compound ~ RF0.6.
aq_cas_01 is high in N,N-DMT
aq_irn_01 and aq_scc_01 are high in 5-MeO-N,N-DMT

 

[Grasshoppers]

I modified the mobile phase to improve separation.

The new mobile phase consists of ethyl acetate, methanol, and 25% aqueous ammonia in a ratio of 13:6:1.

Here are the observed Rf values:

Rf 0.20 - NMT and 5-MeO-NMT
Rf 0.33 - Gramine
Rf 0.50 - N,N-DMT and
Rf 0.55 - 5-MeO-DMT
Rf 0.70 - unknown alkaloid

Have a look!

275nm wet plate

275nm dry plate


Samples aq_tur_03 and aq_scc_03 show particularly high levels of 5-MeO-DMT, while aq_arg_01 and aq_arg_02 are the most potent N,N-DMT-dominant plants we've encountered this year.

Interestingly, aq_sil_02 once again contains that unidentified alkaloid. Should we focus on identifying it, or consider selecting for it in future breeding efforts?