Just look at that thing. Look at it. Wow. Wow. That is a passionflower. There are many like it, but this one is mine. Warning: The leaves of *Passiflora spp.* can produce toxic cyanide compounds. Probably don't consume a significant amount of passionflower leaves if you are pregnant or nursing. But hey... you can still look at it. Just look at it... **amazement... shake head** As always, a research report is linked below and a timestamp for the germination protocol is here. *indicate right* *Passiflora incarnata* is one of over 500 species in the Passiflora genus. About 20 of those are cultivated commercially. Passiflora edulis is probably the one that most people are familiar with. When someone says "Passion Fruit" they almost always mean Passiflora edulis. It is cultivated for the 15-20 ml of delicious and fragrant juice that comes by mashing the fruit. *Passiflora caerulea* is probably the other most commonly cultivated of the genus. A few varieties are grown mostly for their horticultural merits since the fruit is rather bland or undersized. Some hybrids are available as well as grafted nursery plants. Just know that Passiflora is a diverse genus, especially when it comes to germination. Even more, the subject of today's video, incarnata, is one of the rare cold-tolerant species. What works for one will not necessarily work for another and there is a lot of misinformation out there equating the germination procedure of one Passiflora species with another. So why did I choose incarnata out of the many hundreds? Well for starters, this one might grow in my backyard in Kansas. While most Passiflora hail from Central or South America, incarnata is native to North America. This makes these plants a little more cold-hardy than their tropical counterparts. They have been sighted growing wild as far north as zone 3, but more commonly inhabit the southeastern states of zones 7-10. Incarnata survive the cold by sacrificing their above-ground shoot and regrowing in the spring using stored nutrients in the roots and rhizomes. Their regrowth is sudden and punctual leading to the moniker: Maypop. Get it... pop out in May. Any area where the freezing temperatures do not penetrate deep enough to kill the roots can be suitable for outdoor cultivation. If you live in planting zone 7 or more, you might have seen some wild incarnata growing abundantly in disturbed and otherwise barren habitats. The growing region can be extended slightly in protected areas shielded from the worst of the cold by underbrush or terrain. The individual plants of wild stands are likely siblings, having been ehem... deposited... by a large animal (including humans) that consumed the fruit at some point in the past. That is their preferred method of dispersion and propagation. Passiflora can become aggressive in the right conditions and are sometimes considered an agricultural pest. Once established, they are difficult to remove. They can regrow from even the smallest root fragments and spread rapidly through the soil. They are so aggressive that large plantations rarely have to remove weeds from mature plots. If you decide to plant it outside, make sure you want it there. Forever. Removing one will probably require a 1-2 meter deep hole 12 meters in diameter (larger for expanded plants). If you miss even a single 5mm root section, incarnata can and will regenerate. Most herbicides don't work all that well either. It might be easier for **you** to move if you don't want a passionflower anymore. Also, incarnata sometimes produce extrafloral nectaries that attract ants. It's a symbiotic relationship where plants exchange some sugar for an army of ants that ward off herbivorous insects. Don't plant them anywhere that can't handle a couple of ants. Another reason to grow incarnata is for its purported medicinal properties. While edulis may have juice in abundance, each maypop fruit only produces a paltry 1-3ml. Instead, incarnata are grown for the flavonoid and alkaloid contents of their leaves. Now I'm not particularly interested in this potential per se, but I am interested in how incarnata makes these compounds. You see, when I started looking at Passiflora I came across something I had not seen before: some of the fungi and bacteria naturally inhabiting incarnata leaves also produce the same compounds that incarnata is known for. I'll talk more about that in a minute. So a beautiful flower, able to grow outdoors in my location, some amazingly complex and perhaps symbiotic dependent phytochemistry... What's not to love? Well... unlike Coffea arabica, I don't have a lot of seed choices. Incarnata are not produced by the millions of kilograms so I take what I can get from the small online vendors. Large seed companies don't seem to carry incarnata. Perhaps this is due to their self-incompatibility? Something that would make routine seed production a little more difficult. Or maybe the demand isn't high enough. Though I can't understand why. Look at that thing. We can talk more about flowers, fruiting, and fertilization techniques if and when I get some flowering incarnata. Today we are focussing on germination. Online recommendations for incarnata span the whole gamut. Some internet sources would have you believe that it reproduces primarily by rhizome. Some claim that their seeds are recalcitrant and impossible to grow once dry. Some recommend physical scarification or stratification to germinate these "stubborn" seeds. Some don't even distinguish between the obviously different Passiflora species. Certainly some species in the Passiflora genus have recalcitrant seeds and others require scarification. But not incarnata. I believe a lot of these misconceptions arise from the intense similarity between certain passiflora species. The similarity is so strong, that the early literature often equated incarnata and edulis with later researchers admonishing the sloppy identification. If you get a second, read *Dhawan et al. 2001*. It is entertaining to see an author throwing shade *heh shade* in a peer-reviewed journal article. Only intense scrutiny of the flowering structures or microscopic morphology can positively distinguish the two. Alternatively, you could attempt some test pollination to disambiguate an incarnata and an unknown Passiflora. If no seed-set is produced by the cross, or the offspring is sterile, they are likely different species. Though that isn't a perfect method because the plants could be close enough genetically to trigger self-incompatibility mechanisms. Many commercial breeders have been attempting to generate fertile crosses of the cold-hardy incarnata with other high-productivity tropical species without much success. Identification by the simple color of flowers or fruit isn't a good option. Passiflora are naturally quite diverse and they have been subject to the machinations of plant breeders for decades. Even among positively identified incarnata, there exists significant variability in the chemicals produced by the plant. Unfortunately, very little information is available to the amateur ethnobotanist for testing such things beyond, "Eat it and see what happens." Let's hope my seed vendor was careful in **their** identification. I guess we will find out. Still... incarnata must have some environmental cue or cues for germination. This ain't the tropics. Germination at the wrong time in North America can be a death sentence. The most important factor contributing to incarnata germination is temperature, followed closely by light. The lower germination threshold of incarnata is about 25 degrees Celcius. That means that no seeds will germinate if incubated below 25 C. Even among tropical plants, this is unusually high. Similarly, incarnata shows pronounced *photoblasty* though only during a brief window. They have a strong preference for darkness during the first 24-48 hours of germination depending on the temperature. A few minutes of light during the initial stages or any amount after 48 hours, regardless of the quality, won't affect germination rates. In systematic tests, incarnata geminates best at 35 degrees Celcius in the dark. Beyond that, it starts to get a little fuzzy. The few studies on incarnata have used washed and dried seeds or don't state the other conditions used. Many other Passiflora species rely on drying or leaching to break physiological or chemical dormancy, respectively. Indeed some tests have shown that leaching incarnata seeds in tap water for 8 hours or more can increase germination rates, although the evidence is somewhat mixed and confounded by other variables. There seems to be no evidence of *vivipary* or precocious germination in passiflora. So it stands to reason that something is preventing germination inside the fruit. When heat and light conditions are suboptimal, the seeds appear to benefit from other treatments such as leaching, pre-chilling, or gibberellin treatment. Ultimately, however, these treatments rarely enhance germination beyond what high temperature and darkness can do. So it is likely that chemical and physiological dormancy is minimal in incarnata. For me, though, this is irrelevant. My seeds are clean and dry as I expect any mail-order seeds will be. So any chemical inhibitors were probably already removed along with the *aril*. I'd say you can expect around 50% germination over 10-14 days with optimal conditions and good quality seeds. Germination as high as 84% has been noted in the literature for dried incarnata seeds stored at 5 degrees Celcius for 6 months. The refrigerator is probably a good long-term storage solution, if needed. Another variety from that same study and in the same conditions had a maximum germination of 65%. The variation seems to be due to the plant's genotype, fertilization, or harvesting protocol and not from any particular storage condition or seed treatment. This also makes it difficult to compare germination rates between studies since standardized accessions of incarnata are localized at best and outbreeding is required to produce fertile seeds. Also, many studies use seeds the authors collected from whatever wild passiflora are in the area, sometimes without positively identifying the plant as a true incarnata. The wide discrepancy from plant to plant could have also contributed to some of the more exorbitant germination methods recommended online. Germination rates might be predictable from seed characteristics such as weight or color, but this hasn't been tested directly. There is definitely a need for some high-quality germination research on Passiflora. Also, remember that incarnata are self-incompatible. If you would like fruit, you need to have at least two plants preferably with disparate genetics for maximum seed set probability. Entire monoculture plantations of incarnata grown for medicinal purposes can be utterly void of fruit. More plants won't necessarily induce more flower production, though, if that is your goal. The primary pollinators of incarnata, *Xylocopa spp.*, have a range of several kilometers. If you live within a bee's flight to another incarnata, outdoor cultivation of a single plant might yield fruit without any intervention on your part. Manual pollination will be required for indoor plants, however. Typically, my germination protocols start with some sort of disinfection but that is not the case here. Incarnata, perhaps more so than its fruit-bearing counterparts, may benefit from companion microorganisms. At least 204 identifiable species of bacteria and 315 species of fungi have been isolated from incarnata. I don't know if this is usually numerous or just unusually well studied. As I touched on earlier, some of these species when grown separately produce the same flavonoids as incarnata itself. It makes me wonder what contribution endophytes have to the production of other medicinal and aromatic compounds in plants. It is unclear whether these microorganisms acquired the genes by "stealing" them from the host plant or by some sort of convergent mutualism and what contribution they ultimately make to flavonoid production in traditional field cultivation. In the future, high-tech submerged fermentation of these fungi and bacteria has the potential for increased production of these medicinal compounds at a lower cost, both financially and environmentally, compared to whole plant production in a field. Unfortunately, I don't have a bioreactor for plant cell culture... yet. So I'm going to produce these compounds the traditional way: The agronomy way. One thing that is clear with beneficial microorganisms is it helps to start early. Symbiotic organisms are especially helpful during times of stress such as germination and early seedling development. The more difficult the growing conditions, the more plants tend to rely on their symbiotic relationships for assistance. In other words, plants have a *Relative Mycorrhizal Dependency* based on their needs and the environment. If your plants are kept in perfect growing conditions all their life, they may not benefit from inoculation. If, however, your plants experience drought, hydration stress, low nitrogen, or phosphorus at any time, just to name a few adverse conditions, inoculation is something to consider. It's not just the roots, either. The leaves and stems of incarnata support a plethora of microorganisms. Inoculating the seed starting media ensures that the best species are carried with the developing shoot as it grows. Since incarnata's microorganisms supposedly have specific genes related to bioactive molecule production, our inoculation choices are somewhat limited. The "all-purpose mycorrhizal granules" sold online might help with the generalized growth of the plant, but won't necessarily increase the production of the compounds that incarnata are known for. Though any inoculation is probably better than none. Inoculating new incarnata directly with endophytes from established plants that produce the molecules in question abundantly will likely yield the best results. Side note: I don't know why incarnata rhizomes aren't the primary method of mail-order propagation. They have more abundant energy reserves than seeds making them perhaps more tolerant of poor initial planting conditions. They don't suffer from the reproductive viability issues that plague seeds. They have specific, known genetics that would make production more predictable. They come preinoculated with the ideal set of beneficial microorganisms. These seem like great advantages. Perhaps I'm missing something. Anyway, since I don't have access to specific isolates, I'll have to rely on good old-fashioned horizontal gene transfer in generic species. To that end, I looked for a commercial inoculant that had as many of the microorganisms that were commonly associated with incarnata as possible. Genera to look for include: *Gigaspora spp.* *Sphingomonas spp.* *Trichoderma spp.* *Scutellospora spp.* *Aspergillus spp.* *Claroideoglomus spp.* You can look for more specifics in the research report below. Alternatively, you can source these microorganisms directly from the environment by mixing some raw garden soil with your seed starting mix. The best results are likely to come from regions that grow incarnata naturally. Or just use fresh compost or vermicompost. Preferably some that you produce yourself. I must warn you though: Root rot and Fusarium wilt are a problem for incarnata, especially during the winter dieback period. If you use broad-spectrum inoculants like compost or soil, some of these diseases could also transfer. There are no satisfactory chemical controls of wilt and root rot in Passiflora, so preventing the initial infection and keeping the roots sufficiently dry are the only methods of protection. All of this is not to say that you should avoid seed disinfection entirely. Beneficial microorganisms are often transmitted inside the seed itself. Only after germination are they released. Pathogenic species, in contrast, tend to adhere to the seed surface, so disinfecting it shouldn't affect the good ones inside. Similarly, incarnata are relatively fast germinators, so they will have their defenses in place before the pathogens get a chance to take over. I chose not to disinfect my seeds this time, but there is no reason not to try a wash with 10% bleach for a few minutes if you have infection problems. If you are worried, try a test germination with a small subset to determine the cleanliness of your seeds before sterilizing the batch if necessary. Given all of this background information, how should we proceed? I believe leaching to be sufficient for pre-germination treatments. It is called "leaching" because that's what is happening: A large volume of water dissolves away the chemical inhibitors, thereby promoting germination. But it is the same process as what's commonly called seed soaking, perhaps with more water than usual. For my part, I submerged five seeds in 30ml of tap water or about 200:1 by weight. I have no idea if this is the optimal volume of water to sufficiently dissolve the inhibitors or if those inhibitors exist, but the tiny beaker looked cute on camera. So... The beaker was covered in aluminum foil to block out the light. I did this leaching step at room temperature, but it might be beneficial to do this in an incubator to shorten the total germination time. Leach your seeds for 12 to 24 hours. My seeds were soaked for 48 hours because *reasons*, but that shouldn't affect germination. After that, sow your seeds in whatever seed starting mix you prefer. Any depth to 8cm does not affect total emergence. Deeper sowing will protect your seed from the variable hydrology of the surface but it might slow emergence slightly. Similarly, the deep media shields your seeds from light and improves overall germination rates compared to surface sowing. If you do not have a temperature-controlled germinator, I recommend planting your seeds in deep pots and placing them on a sunny windowsill and/or on a seed heating mat. Neither of these methods will result in higher germination than a dedicated incubator, though. Seed mats typically max out around 30 degrees Celcius and the temperature fluctuations of a sunny windowsill are worse than constant warmth but better than nothing. The ideal conditions are a constant 35 degrees Celcius in the dark for at least 24 but preferably 48 hours. Longer for lower temperatures. It is only during this intial period that those special lighting conditions are necessary. So rig whatever apparatus you can for this initial bump. I used the lab-standard filter paper and petri dish method mostly for inspection and visual appeal. A person of the apartment persuasion like me can only have so many incubators, but just like lovers of all things green bringing home the plant they bought on sale: I can fit just one more. Now that's a properly tuned PID controller that doesn't fail during filming! *show stable temperature* And now we wait. *black screen* That's where this script has sat for the last three months, waiting for seeds to germinate. That's three months for a seed that should take less than two weeks. In all that time, I had one germination from three batches of 5 seeds each. I even tried some pretreatment variations. Nothing. There are three scenarios I foresee: One, the seed seller produced infertile seeds either by harvesting immature fruits or attempting to inbreed their mother plants. Two, the seed seller misidentified some other Passiflora. One that requires more elaborate pregermination treatments. Therefore, my protocol is based upon a false premise and doomed to fail. And three, that I am simply incompetent and messed up the germination somehow. Or it's some comination of these possibilities or one I did not forsee. I don't have any more seeds from that seller upon which to test alternative pretreatments. And I'm not buying anything else from them. That's for sure. Though you are likely to hear me complain more about them in the future as I work my way through the other seeds I purchased at the same time. In any case, one seed did germinate so eventually I will be able to identify the species by the flower, though we will have to wait a while for that video. Fortunately, I happened across someone who lives just down the street from me who was very generous with the fruit from their backyard plant. Through a strange set of coincidences involving a somewhat esoteric plant website and some Facebook stalking, I received 8 partially-dehydrated incarnata fruits with hundreds of seeds inside. I have not personally verified that they are incarnata, but given my cold location, it is unlikely that they are anything else. I also bought more incarnata seeds from two other online sellers. Hopefully the different sources yield some decent genetic variation and I look forward to comparing the sets in the future. Regardless of my lackluster germination results, the methods layed out in this video prescribe your best chance of success. After all, nothing I do here is as rigorous as peer-reviewed research, which is what I am attempting to convey to you. I look forward to a time when mail-order medicinal seeds evoke fewer relatively unsuccessful videos such as this one. Until then...