Development and Optimization of a Germination Assay and Long-Term Storage for Cannabis sativa Pollen
Pollen viability and storage is of great interest to cannabis breeders and researchers to maintain desirable germplasm for future use in breeding or for biotechnological and gene editing applications. Here, we report a simple and efficient cryopreservation method for long-term storage of Cannabis sativa pollen. Additionally, the bicellular nature of cannabis pollen was identified using DAPI (4′,6-diamidino-2-phenylindole) staining. A pollen germination assay was developed to assess cannabis pollen viability and used to demonstrate that pollen collected from different principal growth stages exhibited differential longevity. Finally, a simple and efficient method that employs pollen combined with baked whole wheat flour and subsequent desiccation under vacuum was developed for the long-term cryopreservation of C. sativa pollen. Using this method, pollen viability was maintained in liquid nitrogen after four months, suggesting long-term preservation of cannabis pollen.
Cannabis or hemp (Cannabis sativa L.) is an annual, primarily dioecious flowering plant. The center of origin is in Central Asia, and it has been bred for thousands of years for a variety of traits, including fiber, oil, seed and drug use . Cannabis is a diploid plant (2n = 20) and males are characterized by heterogametic chromosomes (XY) with homogametic chromosomes (XX) conferring the female phenotype. Male plants produce flowers containing stamens producing pollen whereas female plants develop ovaries that produce seed following pollination. Female inflorescences are characterized by secretory hairs known as glandular trichomes, which produce a resinous mix of cannabinoids and aromatic compounds that are valued for both medical therapeutics and recreational effects .
Pollen viability is of great interest to breeders and researchers alike. Breeding projects may wish to store pollen for extended periods of time, where high value genetic material may be stored for future use or for biotechnological and gene editing applications that requires a quick and effective method for determining pollen viability [3,4,5]. Fluorescent stains such as fluorescein diacetate (FDA) or fluorochromatic reaction test (FCR) have been previously reported for assessing pollen viability in cannabis [3,4]. Viability is not always correlated with germination, as pollen may retain the ability to metabolize while losing its ability to germinate . To better assess germination, we established a pollen germination assay (PGA) to estimate germination rates. We also adapted a DAPI (4′,6-diamidino-2-phenylindole) stain to visualize pollen pre- and post-germination, and to establish whether Cannabis sativa pollen was a bicellular or tricellular, which to our knowledge has not been reported in the literature. In approximately 30% of angiosperms, pollen is tricellular, with the male gametophyte sexually mature at the time of anthesis . We also used the PGA to test how storage and timing of pollen collection influences germination rates. Pollen germination rates were assessed over a period when stored at 4 °C from males at different stages of floral development. Finally, we developed a simple procedure for the long-term storage of cannabis pollen using desiccation with baked whole wheat flower followed by cryopreservation, which potentially maintains long-term viability of pollen for future use.
2. Results and Discussion
2.1. Optimization of Pollen Germination for PGA
To obtain a representation of the germination profile, a time-lapse of a pollen germination assay (PGA) was evaluated using microscopy. We observed the germination profile for 6 h with 30 min interval. The final germination was calculated after 16 h incubation. Germination started within 30 min with extending pollen tubes clearly visible ( Figure 1 and Video S1).
Representative photographs of cannabis pollen germination profile. Images were taken at 30-min intervals for 6 h with germinating pollen grains indicated by the yellow arrows. Germination started within 30 min with extending pollen tubes clearly visible. Images were acquired using an inverted fluorescent microscope (Zeiss Axio Observer Z1, Germany).
Cannabis pollen readily germinated in the Pollen Germination Media (PGM). PGM was evaluated both as a liquid and a solid media (1% agar). Germination rates were comparable in both media; however, pollen tubes were not as easily imaged under the microscope when germinated on solid agar medium (data not shown). For this reason, we opted for performing the PGA using liquid media. Of the different concentrations of pollen tested, 0.1 mg/mL provided the clearest imaging of germination, as higher concentrations resulted in crowding in the test well that reduced visibility ( Figure 2 ). Additionally, in the highest density treatment, germination was adversely affected and made it difficult to accurately quantify germination percentage ( Figure 2 ).
Optimization of the Pollen Germination Assay. Cannabis pollen germination in PGM at concentrations of 0.1, 1 and 10 mg/mL. Images were acquired after 16 h using an inverted fluorescent microscope (Zeiss Axio Observer Z1, Germany).
2.2. Pollen Collected at Different Principal Growth Stages Exhibits Different Longevity
To establish how cannabis pollen germination rates change over time, we tested the pollen in a pollen germination assay after storage at 4 °C. Because pollen collected from different principal growth stages may affect germination rates, we collected pollen from male flowers at four different points during floral development to cover the entirety of anthesis (Figure S1).
We compared the loss of viability of cannabis pollen collected from the four different points during flower development over the course of 21 days. The rate of germination at T0 was 33% for Early (62), 46% for Mid (64), 50% for Mid-Late (65) and 41% for Late (64) stage ( Figure 3 ). All stages lost viability after only one week at 4 °C storage, except Mid (64) ( Figure 3 ). After 21 days storage at 4 °C, pollen collected from Early (62), Mid-Late (65) and Late (67) stages, lost their viability (approached 0% germination). However, pollen collected from the Mid flowering stage (64) retained viability the longest with 22% of pollen grains successfully germinated after 21 days storage at 4 °C ( Figure 3 ). This suggested that an optimal growth stage for pollen collection is around the developmental stage (64), whereas the loss of pollen viability may begin while the pollen is still present in the anthers. Pollen collected earlier, at developmental stage 62 may not have fully matured, resulting in a lower germination percentage ( Figure 3 ).
Loss of pollen viability over time. Pollen was harvested from plants at four different developmental stages then stored at 4 °C for one to three weeks. Viability was determined via pollen germination assay. Data were shown as mean ± SE (n = 9).
2.3. DAPI Staining Revealed Bicellular Nature of Cannabis Pollen
While the fluorescein diacetate (FDA) stain is routinely used for viability tests, it is not ideal for visualizing the nuclei in pollen cells. In order to establish whether cannabis pollen was bicellular or tricellular, we performed a DAPI stain on germinating cannabis pollen. Prior to pollen tube germination, the brighter, more compact sperm nucleus and the diffuse vegetative nucleus were visible ( Figure 4 A,B). The brighter staining in the sperm nucleus represents the more condensed state of chromatin compared to the more transcriptionally active vegetative nucleus. Following pollen tube germination, both sperm nuclei are clearly visible as they descend the pollen tube ( Figure 4 C). This suggests that cannabis releases sexually immature pollen grains, with the second mitosis event occurring after pollen tube germination.
Visualization of cannabis pollen at different stages of germination using DAPI staining. Both the sperm (SN) and the vegetative nuclei (VN) are visible at the bicellular stage prior to pollen tube germination (A, B). Image (C) represents a germinated cannabis pollen cell.
2.4. Development of a Cryopreservation Method for Cannabis Pollen
Pollen cryopreservation has been employed in a variety of agriculturally and medicinally important plant species for the preservation of elite germplasm. Numerous studies have reported the data on pollen viability under various storage conditions [5,7]. While the interaction between pollen water content and viability is complex, it is understood that optimum water content is necessary for longevity . Generally, longevity is increased by lowering the temperature and moisture content. Some reports indicate a moisture optima of 15%, while higher water concentrations (above 30%) may result in rapid deterioration during cryopreservation . Liquid Nitrogen (LN; −196 °C) is routinely used for cryogenic storage, as it is relatively cheap, safe and maintains a temperature where enzymatic and chemical reactions do not cause biological deterioration . Cannabis pollen stored in LN without prior desiccation failed to germinate (Figure S2). Pollen cells with high moisture levels do not survive cryogenic storage, presumably due to intracellular ice formation . Therefore, pollen cells need to be dried within a range where no freezable water exists without succumbing to desiccation injury. For pollen desiccation, we tested a vacuum desiccation at pressures of 5, 15 or 25 kPa for either 20 or 40 min. When pollen was desiccated prior to storage in LN, it failed to germinate (Figure S2), suggesting that desiccation alone may not be sufficient for pollen viability during cryopreservation. Therefore, in addition to desiccation, we also tested cellular cryoprotectants, such as DMSO and glycerol that have been reported to improve cell survival after cryogenic storage . Desiccated cannabis pollen combined with a 10%, 20%, 30% or 60% DMSO or glycerol solution prior to being stored in LN for 24 h exhibited 0% germination (Figure S2).
Baked wheat flour has been previously suggested as a possible cryoprotectant for long term pollen storage . To test whether baked wheat flour can be used as a cryoprotectant for cannabis pollen, cannabis pollen was desiccated and combined with baked wheat flour. Vacuum desiccation at a lower pressure of 5 kPa for the longest interval for 40 min, resulted in the highest germination rate after storage in LN after 24 h ( Figure 5 ). Pollen germination did not occur at higher pressures, as the cells may have been compromised during the drying process. This treatment was used for subsequent preservation experiments where the GLM test results indicated no significant differences in germination rate between 24 h LN stored pollen and the non-LN control pollen (p > 0.05) that was subjected to the same desiccation protocol and combined with whole wheat flour ( Figure 6 ). Desiccation itself caused approximately 50% reduction in germination as compared to untreated freshly harvested pollen ( Figure 3 and Figure 6 ). Desiccated cannabis pollen combined with baked wheat flour was kept in LN for four months to test long term storage. The GLM test results indicated that there was no significant difference observed as compared to non-LN control and 24 h LN stored pollen (p > 0.05) ( Figure 6 ), suggesting long term storage is a possibility under appropriate conditions. To confirm in planta viability of the treated cannabis pollen, the pollen/wheat flour mix was removed from LN and applied to flowering female cannabis plants. The pollination resulted in successful seed formation in all the flowers receiving treated pollen. Once the female had finished flowering, the flower material was collected and processed for seeds. Seed number, size and morphology from the cryopreserved pollen were similar to those obtained using untreated fresh pollen (Figure S3). Collected seeds were germinated to ensure viability, with no abnormalities noted.
Representative photographs from pollen germination assay (PGA) of pollen stored for 24 h in liquid nitrogen (LN). Desiccated cannabis pollen mixed with 1:10 wheat flour and stored in liquid nitrogen (LN). Non-LN control (control was subjected to the same desiccation combined with whole baked wheat flour). Pollen flour mix was diluted to 0.1 mg/mL in PGM and used for PGA.
How to Take Time-Lapse Pictures of Your Plants Growing
Plant life comes at you fast; before you know it that little sprout is a full grown monster plant, desperate for a bigger pot. If you’re a green thumb type who’s fascinated by your plant’s progress, here is the perfect way to document every tender unfurling.
Artist and programmer Nicole He set up a camera powered by a Raspberry Pi computer to document the slow growth of her fiddle-leaf fig plant. She programmed the computer to post a picture everyday to Twitter under the handle @grow_slow , in case anyone else wanted to follow its development. After two years, He compiled them all into a little video of “growth and movement”:
On her website, He writes that the project is a “meditation on nature and technology,” and explains that the webcam takes a picture of her plant every morning at 10:17 am:
Often, I’d be going about my life, happen to take a glance at my plant and think, “huh, when did it grow so fast?” I found it easy to forget that plants – being alive and all – are not just household decorations, but organisms that move and change and grow.
The internet is fast, but plants are slow. This project is an experiment in combining those two things. @grow_slow follows the philosophy of the Slow Web . Any one picture might be mundane by itself, but as a collection, they become interesting over the course of time.
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He also provides a step-by-step how to for setting up your own technology meditation—or if it’s not that deep, a way to take daily pictures of your plant.
What You’ll Need
He uses a Logitech HD Webcam C310, which she connects via USB to a Raspberry Pi, an external monitor and keyboard, and some sort of wifi connection if you also want to send pics to the Internet. She uses a Linux utility called a cron job, which is basically a scheduler, with a Python script. Oh, you also need the plant.
Set Up Your Pi
The Always-Up-to-Date Guide to Setting Up Your Raspberry Pi
So, you finally picked up a Raspberry Pi and it’s sitting on your desk, waiting for you to do…
He recommends setting up SSH access from another computer, because it may not always be convenient to connect a monitor, keyboard, and mouse to the webcam in charge of documenting planty. But for beginners with the Raspberry Pi, it’s helpful to have those things.
Set The Date For Your Timer
You presumably want to set a specific time on a daily basis, rather than getting five hundred pictures in an hour. You have to make sure your Pi is set to the right date. Sometimes it’ll set automatically, so type in “date” to check. If it’s wrong, type “tzselect,” and it’ll walk you through selecting the correct timezone.
Connect The Webcam
The webcam should connect vis USB to the Pi, but to work it, He offers this code:
In order to get the webcam working with the Pi, we’re going to install a package called fswebcam :
sudo apt-get install fswebcam
With the USB camera plugged in, we can very easily take photos via the command line:
Give It Social Media (Life)
You need a Twitter handle to send these pics to if you want to follow in He’s footsteps. She used a Python script to run once a day and tweet the plant photos from her webcam, which requires a package called Twython, and the cron task schedular. Her instructions are fairly involved (but still totally doable!) and you should check them out here . And remember, this is a project He wants to share so people can bend it to their own needs.
“You can change the frequency of the photos, or give the photo names a timestamp, or upload each photo onto a server, or tweet words as well as photos, or instead of taking photos of your plant take photos of your piano, or window, or bed, or aquarium, or whatever,” she writes.
From Seed to Harvest: A guide to growing strawberries.
Strawberries are frequently grown from bareroot, which are seeds that have developed and grown into roots, which are then easier to start in a garden. However, it is possible to start strawberry plants from seeds as well, it just takes a longer time and certain steps must be followed to ensure the best growth. Growing strawberries from seed allows the garden to have a wider variety of which types of strawberries they wish to grow.
Although bareroot strawberries can be planted at any time, strawberry seeds must be started indoors to ensure they are ready to go when it comes to their normal growing season. To ensure strawberry seeds will grow when you plant them, you must first stratify them. To do this, place the strawberry seeds in an envelope or sealed plastic bag and store them in the refrigerator for a month. Since strawberry seeds must be planted by February to be ready for harvest, begin the stratification process in January. Once the month has passed, remove the seeds but leave them sealed overnight. Open them the next day.
Cold stratification is required for certain seeds to sprout properly. Cold stratification is a process in which seed dormancy is broken by mimicking the natural conditions a seed might go through.
After you have gone through the stratification process, plant the seeds 1/2 inch deep in a container filled with sterile seed-starting mix. Press the seeds into the surface but do not cover them, because in addition to cold stratification, strawberry seeds require light to germinate. Seeds will germinate in one to six weeks. Six weeks after the seeds germinate, transplant them into bigger, individual pots. In another six weeks, your strawberry seedlings are ready to plant outside.
Harden off your strawberry seedlings by placing them outdoors in protected areas for half hour increments. Gradually increase the time the plants spend outside. Once they are hardened off, transplant the seedlings into the garden. Space them two feet apart and be sure to plant them in well-drained and acidic soil. Strawberries require full sun to grow. Water the strawberries at least 1 inch a week during their growing season. In addition to growing in a strawberry patch in the garden, strawberry plants also can grow in pots, strawberry towers, raised beds and more. The roots don’t grow deep, meaning the strawberry plant doesn’t need deep soil to grow in.
Once the growing season is over, cut the foliage back to 1 inch. Mulch over the plants with 4 inches worth of straw, pine needles or another type of organic material. Remove the mulch in the spring.
Strawberries are ready to harvest as soon as they turn red. They should be slightly firm to the touch, but if strawberries are too mushy, they can be used to make jellies and jams. Strawberries likely won’t be ready to harvest the first year you plant the seeds because many times, gardeners pinch off the blooms to direct energy into the plant itself. This allows the strawberry plant to direct all of its energy to its roots and leaves, which will need to be strong to produce fruit in the second year of growth.
When they are ready to harvest, cut the berry off at the stem. Do not pull the berry from the stem to harvest it. Unwashed strawberries can be stored in the refrigerator for three to five days.
What strawberries crave:
Begin the fertilizing process when seedlings are still indoors. Start fertilizing with liquid kelp every two weeks for the first month of growth. Use only one tablespoon of kelp per gallon of water to fertilize. After the first month, double the fertilizer strength. Prior to transplanting the strawberries outside, mix ¼ cup of 5-5-5 all-purpose organic fertilizer into the holes meant for each transplant. Water after transplanting and mulch with straw. The last time you will feed the strawberry plants will be after you harvest. At this time, add compost into the soil around the plant. Strawberries are perennials, which means they return each year, and taking care of the soil will allow the strawberries to grow back even stronger than before.
Where to buy strawberry plants:
You can find a wide variety of both strawberry seeds and plants at Urban Farmer.
How to: Grow Strawberry Roots
Fresh, sweet strawverries from a homegrown berry patch add to the deliciousness of summer, and gardeners wanting to start their own berry patch usually purchase bare root strawberry plants. Bare roots are the roots of a strawberry plant that are dormant and not planted in soil. To ensure the plant wakes up from its dormant state, bare root strawberry plants must be planted properly.
How to plant bare roots:
It is time to plant bare root strawberry plants once all danger of frost has passed. Prepare the strawberry patch by choosing an area in full sun that has good drainage. Test the soil of the chosen area and make sure the soil pH is between 5.8 and 6.5. Amend the soil with 3 inches of compost and 1 pound of 10-10-10 fertilizer per every 100 square feet of the strawberry patch. Before planting, trim the bare roots to a length of 6 inches. Soak the bare roots in a bucket of water for 20 minutes. Only soak the roots, do not submerge the entire plant. This breaks the dormancy cycle by rehydrating the roots.
Dig holes in the bed the depth of the bare roots and two times as wide. Spread out the roots and keep the crown slightly above the level of the soil. If soil covers the crown, the crown can rot and not produce any plants. Space roots 18 inches apart in rows set 3 inches apart. Water the plants immediately after planting them and lay 2 inches of mulch over the newly planted roots. Water the bed up to 2 inches each week.
Caring for bare root plants:
Once the strawberries wake up and begin to sprout, you can add a side dressing of fertilizer and again after the harvest has ended. After a few seasons of growing the strawberries, the plants will begin to grow runners with baby strawberry plants. Guide the plants across the row as they form and once it is long enough to reach all the way across, encourage the baby plant to grow by weighing down the stem with some weighed object, like a small rock. Every third year you can remove the mother plants and the baby plants will begin to produce, which makes the strawberry patch self-regenerating.
Types of strawberries to choose from:
There are three types of strawberry plants. The most popular strawberry plants are the June-bearing varieties, which produce fruit in May and June. These plants only produce fruit once a year, which makes them good for creating jams and jellies.
There also are ever-bearing strawberry plants, which produce fruit twice a year. Fruit isn’t produced until the second year after planting, and they are ready to harvest in mid-summer and in the fall.
Day Neutral strawberries produce fruit all season long. This is the best variety if gardeners are growing strawberries to eat them fresh.