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Inuence of dierent priming
treatments on germination
potential and seedling
establishment of four important
hemp (Cannabis sativa L.) cultivars
Saba Latif1, Rahmatullah Qureshi1, Abdul Rauf2, NoshinIlyas1, Qaiser Hussain3,
Syed Sabir Hussain Shah1, Sabith Rehman4, Arshad Mahmood Khan5, Nasrullah Khan6,
Mostafa A. Abdel-Maksoud7, Abdul Malik8, Sabiha Fatima9 & Bushra Hafeez Kiani10
Medicinal hemp (Cannabis sativa L.) is gaining popularity due to its natural products with potential
therapeutic properties, and its cultivation has recently been initiated in developing countries such as
Turkey, Iran, India, and Pakistan. The crop is primarily grown using feminized seeds or popular varieties
to achieve higher cannabidiol (CBD) yields, with seeds costing approximately $5–10 each. However,
limited knowledge exists on hemp seed germination under dierent seed priming techniques and
variations in seedling growth parameters such as root and shoot lengths, as well as the inuence of in
vitro germination and growth in soil and cocopeat media. This study hypothesizes that various seed
priming methods could signicantly enhance hemp seed germination and dierent growth media
may distinctly impact seedling growth of the tested cultivars. To test this, the study evaluated four
cultivars/chemovars (viz., Fedora-17, Finola, CBD Pink Kush, and a local variety, Gulistan) under ve
seed priming treatments: indole butyric acid (IBA), moringa leaf extract, KNO3, hydro-priming, and a
control (no priming). A randomized complete block design (RCBD) was used, and experimental data (in
triplicate) were analyzed through analysis of variance (ANOVA) using a time-to-event approach. The
ndings revealed that IBA signicantly (p < 0.05) improved seed germination rates, with CBD Pink Kush
achieving the highest germination response (83%) among the tested cultivars. Furthermore, cocopeat
demonstrated superior performance as a seedling growth medium across all cultivars, indicating its
potential for hemp cultivation practices. This pioneering study provides a baseline for understanding
hemp seed germination and seedling growth under dierent priming and growth media conditions,
oering valuable insights for future research aimed at industrial and medicinal hemp production.
Keywords Hemp cultivars, Seed priming, Germination, Seedling vigor, Cannabis sativa
Hemp (Cannabis sativa L.) is an annual and dioecious plant belonging to the Cannabaceae family. is plant
has been known for thousands of years due to its sustainable use for a variety of purposes. Hemp has garnered
increased interest due to its potential use as medicine and the availability of non-psychotropic cultivars1. Several
European and Asian countries have also established legal cultivation of hemp with THC levels below 0.3%2.
1Department of Botany, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Rawalpindi 46300, Pakistan.
2Department of Zoology, Faculty of Science, University of Azad Jammu and Kashmir, Muzaarabad, Pakistan.
3Department of Environmental and Soil Sciences, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi,
Rawalpindi 46300, Pakistan. 4Department of Botany, Hazara University Mansehra, Mansehra, KPK, Pakistan.
5Department of Botany, Government Hashmat Ali Islamia Associate College Rawalpindi, Rawalpindi 46300,
Pakistan. 6Department of Botany, University of Malakand, Chakdara, P.O. Box 18800, Dir Lower, KPK, Pakistan.
7Botany and Microbiology department, College of Science, King Saud University, Riyadh, Saudi Arabia.
8Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. 9Department
of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia.
10artment of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609, USA. email:
rahmatullahq@uaar.edu.pk
OPEN
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Medicinal hemp has gained signicant attention in recent years, particularly in developing countries such as
Turkey, India, and Pakistan. is surge in interest is attributed to the plant’s diverse applications and the growing
demand for cannabinoids, which are primarily derived from hemp seeds and owers3. However, the cultivation
of hemp, especially for medicinal purposes, presents unique challenges, including the high cost associated with
feminized seeds, which can be as much as $20 per seed.
Optimizing the germination process of hemp seeds is critical to achieving maximum yield and eciency
in cultivation. Seed germination is a complex process inuenced by various factors4. Among them, the choice
of growth media and seed priming techniques play a crucial role in determining germination success and
subsequent seedling growth5. Traditionally, hemp has been grown in Europe for its ber; however, there is now
a growing interest in its diverse applications in Europe and other countries including Pakistan6. Industrial hemp
cultivars produce numerous secondary metabolites, including avonoids, terpenes, and cannabinoids, which are
predominantly found in female owers and can be extracted for pharmaceutical purposes7.
Hemp is renowned for its cannabinoids, particularly the non-psychoactive cannabidiol (CBD), known for its
medicinal properties, and the psychoactive tetrahydrocannabinol (THC). Certain hemp cultivars are specically
bred to be rich in CBD, and an optimized CBD-to-THC ratio enhances their therapeutic potential, making them
ideal for medical applications8. e commercial acceptance of hemp products, both as food and medicine, has
signicantly increased in recent years. However, there remains a lack of comprehensive agronomic information
on hemp cultivation, particularly regarding germination techniques and optimal cultivation practices, which
limits the crop’s potential in various industries9.
Monoecious dual-purpose cultivars are considered the best choice for hemp cultivation, particularly in
Europe, as they enable simultaneous ber and seed production. While Cannabis sativa L. is naturally a dioecious,
modern breeding has produced monoecious cultivars. Monoecious cultivars, which oer higher inorescence
and seed yields compared to dioecious cultivars, have facilitated multiple uses of the crop and improved its
sustainability10. e development of low-THC cultivars has furthered their utility for extracting secondary
metabolites, enhancing their value as dual-purpose crops11. Breeders use selective cross-breeding to enhance
CBD production while minimizing THC levels12. From an agronomic point of view, hemp has been promoted as
a high-yielding crop with low-input cultivation techniques. However, experimental results conrm that special
attention needs to be paid to the germination of hemp, principally medicinal cannabis.
e germination potential of seeds, a critical stage in plant development, can be signicantly inuenced by the
growth medium13. Soil, cocopeat, and hydroponics environments oer distinct conditions for seed germination,
impacting the speed and vigor of the process14. Soil, a traditional medium, provides natural nutrients and a
familiar substrate for seeds, resulting in a slower but oen robust germination process15. Coco peat, on the other
hand, oers a more controlled and nutrient-rich environment, oen leading to faster initial germination16. In
vitro conditions oer precise control over factors such as temperature and humidity, which can signicantly
enhance germination rates. Evaluating germination potentials across various media is essential for optimizing
seedling growth and improving agricultural practices for better crop productivity. Seeds have been subjected to
a variety of research from primitive times. Most of the economically important crops are grown from seeds, so it
is necessary to learn about their germination physiology17. Seed germination is the process by which the embryo
found in the seed develops into a shoot from its plumule and root from its radicle part. Seeds absorb water, which
swells the inactive tissues and starts cell division18. Seeds are heterogeneous storage reserves with a wide array of
storage compounds that include various soluble carbohydrates, starch polymers, storage proteins, and lipids19.
According to physiologists, germination can be dened as the emergence of the radicle via the seed coat, but seed
analysts dened the process of seed germination as the emergence and development of essential structures from
embryos that develop into plants under suitable conditions, while some other botanists dened seed germination
as the emergence and growth of embryos in young plants by the rapture of the seed coat20. e process of seed
germination can be divided into several steps that include the activation of enzymes by imbibition of water, the
development of the radicle to root for absorption of water from the soil, and the development of the plumule
to shoot for photosynthesis21. Seed germination is an essential phenomenon for the survival of plant species.
Dierent environmental conditions, like temperature, moisture, soil, etc., aect the seed germination process4,22.
e study by Toklu23 demonstrated that seed priming with Indole Butyric Acid (IBA) resulted in the highest
germination rates and the longest root development among all treatments. Additionally, IBA priming enhanced
resistance to salinity, making it an eective method for improving seed germination and seedling vigor under
challenging environmental conditions. Another study reported that increasing concentration of IBA priming
resulted in increase of the sprouting and number of leaves per sprout signicantly24. One of the most commonly
used priming techniques is hydro-priming. is process triggers activities linked with the initial stages of
germination25. e experiment conducted by Begum, et al.26 showed that seed priming with Moringa oleifera
extract resulted in a signicant increase in shoot and root length. Moringa leaf extract also increases the number
of secondary roots in hybrid maize seeds. Results by Amooaghaie and Nikzad27 showed that priming with KNO3
media increased enzyme nitrate reductase, which in turn increased the synthesis of nitric oxide, which promoted
speedy germination in tomatoes (Solanum lycopersicum L.).
Fedora-17, a monoecious hemp cultivar, is the result of breeding eorts in Ukraine and France. Renowned
for its early owering, it is specically selected for its adaptability to diverse environments. is cultivar typically
produces 6–8 tonnes of dry biomass per hectare, with seed yields ranging from 0.8 to 1 tonne per hectare, and
a seed oil content of 28–30%, making it a valuable choice for both industrial and agricultural applications28.
Finola is a short, early-owering dioecious hemp cultivar characterized by limited branching and ne bers
resembling ax rather than traditional hemp. Introduced to Canada in 1998, it was ocially included in
Finland’s plant cultivar list in February 2003 and later published in the EU Common Catalogue in August 2003.
Experimental trials in Eastern Finland reported an average seed yield of approximately 1.7 metric tons per
hectare. Finola has been successfully cultivated in Canada, Australia, several European countries, and New
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Zealand, highlighting its global adaptability and agricultural value29. CBD Pink Kush, a feminized hemp cultivar
developed by Seedsman Seeds in Europe, is renowned for its high CBD content, reaching nearly 20%. is
cultivar is specically cultivated for medicinal applications due to its potent therapeutic properties. Gulistan,
on the other hand, is a dioecious hemp cultivar originating from Gulistan, Baluchistan, Pakistan. It is being
utilized for the rst time in our experiment, oering an opportunity to explore its potential and adaptability for
research and cultivation purposes. ere is limited research on the impact of seed priming and growth media on
hemp cultivation. Specically, information about the inuence of dierent priming agents and growing media
on germination potential and seedling vigor in hemp is sporadic. is study aimed to evaluate the eects of
priming agents and growth media on the agronomic and quality attributes of hemp. e research investigated
the impact of four priming agents—Indole Butyric Acid (IBA), Moringa leaf extract, KNO3, and hydro-priming
on the germination potential of four hemp cultivars: Fedora-17, CBD Pink Kush, Finola, and Gulistan. Among
these, Gulistan is a local cultivar, while the others are imported from Europe. Additionally, the study assessed
seedling vigor using three dierent growth media: hydroponics with liquid fertilizer, cocopeat, and soil. is
comprehensive investigation, the rst of its kind, oers valuable insights into the seed germination and seedling
growth of medicinal hemp cultivars. e ndings are expected to guide the selection of optimized priming
techniques and growth media for industrial and medicinal hemp cultivation, contributing to enhanced crop
performance and quality.
Materials and methods
Plant material
Four hemp cultivars, namely Fedora-17, CBD Pink Kush, Finola, and Gulistan (Fig.1) were selected for the
experiment (Table1). Seeds of Fedora-17 and Finola cultivars were obtained from the Canapuglia (URL: h t t p s : /
/ c a n a p u g l i a . i t / e n / c o l l e c t i o n s / a l l ) via online shopping. CBD Pink Kush seeds, a feminized hemp cultivar known
for its high CBD content, were sourced from the Cannabis Seed Bank of Seedsman in the United States of
America. is cultivar is renowned for its medicinal properties and nearly 20% CBD concentration. In addition
to the imported cultivar, a native variety of Cannabis sativa was collected from Gulistan, Baluchistan, Pakistan.
is local cultivar has signicant multipurpose applications in the socio-economic and cultural practices of the
Fig. 1. Growth of four medicinal hemp cultivars in cocopeat: (A) Gulistan; (B) Fedora-17; (C) Finola; (D)
CBD Pink Kush.
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region’s communities. In recognition of its provenance and utility, Prof. Dr. Rahmatullah Qureshi, an esteemed
taxonomist, named the cultivar ‘Gulistan.’ To prepare the seeds for experimentation, they were carefully dried
to maintain moisture content of 12–15% and subsequently stored under refrigeration to preserve their viability.
is meticulous preservation ensures seed quality and reliability for future research and cultivation trials.
Experimental setup
e research was conducted in the Research Laboratory of the Department of Botany, Pir Mehr Ali Shah Arid
Agriculture University, Rawalpindi, Pakistan, and the Institute for Hydroponics Agriculture, Rawat, Islamabad,
Pakistan, from September 2022 to October 2023. For the hemp seed germination experiment involving the
selected cultivars, all glassware and apparatus were sterilized using an autoclave to ensure a contamination-
free environment. e experiment was initiated under carefully controlled conditions, with temperature,
moisture levels, and light exposure meticulously calibrated to maximize germination eciency and ensure the
reproducibility of results. ese parameters were adjusted to reect the specic requirements of each hemp
cultivar, thereby providing an optimal environment for studying germination potential and seedling vigor.
Seeds priming
Prior to seed priming, all the seeds of dierent cultivars were soaked in a solution comprising 10% hydrogen
peroxide (H2O2) and 1% sodium hypochlorite for a ve minutes. Subsequently, the seeds underwent triple
rinsing with distilled water as part of the seed priming treatment. A total of one hundred and twenty (120)
randomly selected apparently healthy looking seeds of each cultivar were treated with solutions of Indole butyric
acid (IBA; 1000ppm), Moringa leaf crude extract (2% in distilled water), KNO3 (4.5% in distilled water), and
hydro-priming (with distilled water) for 24h at room temperature31–33. Similarly, the same numbers of un-
primed seeds of each cultivar were used as a control. Subsequently, all the seeds were transferred in glass petri
plates (150 × 15mm) having lter paper under ambient environment (at about 25–30°C temperature). A total
of ten seeds were transferred to each petri plate. An almost equal amount of water (0.05ml) was applied to each
seed daily and monitoring was done daily to check the germination until no further improvement was noticed
for three consecutive days. Seeds were considered germinated when radicle emerged up to length of 2mm. All
the treatments were carried out in triplicates.
Preparation of Moringa leaf crude extract
Moringa oleifera leaves were collected from the main campus of the Pir Mehr Ali Shah Arid Agriculture
University Rawalpindi, and dried under shade till achieving a consistent weight. A 200g of Moringa leaves were
ground to a ne powder with a grain size of 1mm. e same was mixed in 1L of 80% ethyl alcohol, stirred daily
for three days (72h) under room temperature conditions. Subsequently, the extract was ltered twice through
Whatman No. 1 lter paper, and subjected to a rotary evaporator (40°C temperature and reduced pressure) to
evaporate the alcohol till achieving a consistent weight. Finally, the crude extract was scrapped and stored at
-10°C temperature until further use34–36.
Germination potential
e germination potential of hemp cultivars was assessed by recording various germination parameters including
germination count, germination percentage, root length, shoot length, and seedling vigor index37.
Germination percentage
Germination percentage of seeds was calculated according to the following equation:
Germination Percentage (GP)=
Gf
n×100
Where Gf is the total number of germinated seeds at the end of the experiment and n is the total number of seeds
used in the test38.
Germination rate
e formula for seed germination rate was following:
GR =
(n
N)×100
Where GR represents to seed germination rate, N is total number of seeds used in the experiment and n is the
number of germinated seeds39.
Abbreviation Cultivar Sexual type Origin CBD contents THC contents Fibre Contents Reference
ECV-01 Fedora-17 Monoecious France 2–3% 0.2% 8–10 t/h 30
ECV-02 CBD Pink Kush Feminized North America 17–21% 0.1% No data
ECV-03 Finola Dioecious Finland 2–6% 0.2% 5-7t/h 30
ECV-04 Gulistan Dioecious Pakistan No dat a No data No data Named by Prof. Dr. Rahmatullah Qureshi
Tab le 1. List of origin, sexual type and other important characteristics of tested cultivars.
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Length of seedlings
e lengths of the roots and shoots of the seedlings were measured using a ruler, with measurements recorded
in centimeters (cm).
Seedling Vigor Index
e seedling vigor index (SVI) was estimated according to40 using the following formula:
Seedling vigor index (SVI)=(RL +SL)×(GP)
Where RL is root length (cm), SL is shoot length (cm) and GP is germination percentage. e data of root and
shoot lengths (in cm) of the seedling was measured to determine the seedling vigor index.
Growing media eects
Based on the results of the rst phase of the experiment, which identied the optimal seed priming method
with the most signicant results, the inuence of three dierent growing media on germination and seedling
growth was evaluated. In the second phase of the experiment, the optimal seed priming method was applied
to all the selected hemp cultivars. Germination and seedling growth were tested using the following growing
media: hydroponics with liquid fertilizer (FloraGro from the Flora Series at a concentration of 2ml/L of distilled
water), cocopeat, and local soil. e experiments were conducted under ambient environmental conditions at
the Hydroponic Agriculture facility in Rawat, Islamabad, Pakistan. ese conditions were maintained to assess
the eects of each growing medium on the germination potential and early growth stages of the hemp cultivars.
Statistical analysis
e experimental design was based on a Randomized Completely Block Design (RCBD). e raw data was
stored in spreadsheets. e data analysis was done using linear regression analysis and time-to-event analysis
also known as survival or reliability analysis. e later ones are communicated as more advantageous than the
others41. e GerminaR and germination metrics packages were employed in the R statistical program42 for data
analysis. A total of ten germination indices including germinated seed count, germination seed percentage,
mean germination time, mean germination rate, germination speed, germination uncertainty, germination
synchronization index, variance of the mean germination time, standard deviation of the mean germination
time, and coecient of variance of the mean germination time were recorded37. is quantitative data was
further tested using ANOVA and Tukey’s (HSD) as a post-hoc test when appropriate43,44 to determine the
signicance of seed germination responses for considered cultivars and priming methods.
Results
Germination potential
e germination potential of hemp cultivars was evaluated by measuring various germination parameters such
as germination count, germination percentage, root length, shoot length, and seedling vigor index. e results of
recorded germination indices for the tested hemp cultivars were statistically signicant (p < 0.05) when tested for
seed priming and growth media factors. e results of the germination experiment are shown in Fig.2.
Germination count
e germination counts were recorded over eight days till achieving a consistent response for all hemp cultivars
seeds subjected to dierent priming treatments (Figs.3 and 4). On the rst day, one seed each germinated
for both IBA and Moringa treatments. On the second day, the germination counts were as follows: KNO3
(three seeds), un-primed (three seeds), water (three seeds), and Moringa (two seeds). Moving to the third day,
germination counts were observed to be ve seeds for both un-primed and IBA treatments, four seeds for water,
and three seeds for Moringa. On the fourth day, germination counts were as follows: IBA (six seeds), un-primed
(six seeds), and water (ve seeds). Finally, on the h day, germination counts were noted for un-primed (seven
seeds), IBA (seven seeds), and KNO3 (ve seeds). Maximum germination was observed when seeds were primed
with 1000ppm conc. of IBA followed by un-primed seeds and hydro-priming. Minimum germination was
observed with Moringa extract (Fig.3A-C).
Germination percentage
e percent germination was found directly proportional to the time period. e hydro-priming of seeds
resulted in 15% germination on the rst day; whereas, other treatments did not show any response. On the
2nd day, IBA showed 40% germination, followed by hydro-primed (35%), un-primed (34%), KNO3 (27%), and
Moringa (15%). e germination percentage was 59% in IBA, 56% in un-primed, 40% in hydro-primed, 38.5%
in KNO3, and 24% in Moringa on the third day. On the 4th day, the germination was increased up to 60% in IBA
and Un-primed seeds, followed by hydro-primed (42%), KNO3 (39%), and Moringa (25%) (Fig.4A–C).
e seed germination percentage of all the tested cultivars in terms of priming in dierent media is shown
in (Fig.4A–C). According to the results, maximum seed germination was observed in un-primed and IBA. e
germination rates of the hydro-primed and KNO3-treated seeds were comparable. Minimum seed germination
percentage was observed in Moringa extract. e maximum germination was shown by CBD Pink Kush and
Fedora-17 in IBA, followed by un-primed seeds (Table2). e optimum germination of Finola was calculated
for hydro-primed and IBA (80% each). e results of Gulistan were dierent from the rest of the cultivars. e
maximum germination was observed in un-primed treatment, followed by both IBA and KNO3, hydro-primed
and Moringa. e native variety Gulistan showed best results in un-primed treatments due to its adaptation to
local environmental conditions (Fig.5A, B).
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Germination time
e hydro-primed was the best medium for all the cultivars for germination time. e seeds of Fedora-17, CBD
Pink Kush, and Gulistan were germinated within 2 days of treatment; whereas, Finola expressed germination
on the third day in hydro-priming. e seeds of all the cultivars were germinated within 3 days of treatment
with IBA. e un-primed seeds of CBD Pink Kush, and Finola were germinated on the 4th day; whereas, the
germination of Finola was observed until the 5th day while treated with KNO3. Overall, Gulistan exhibited the
fastest germination; whereas, there was a slight dierence in germination rate in Fedora-17 and CBD Pink Kush.
ere was the lowest germination rate in Finola (Fig.6A, B).
Germination rate
All the four cultivars expressed the mean maximum germination rate in hydro-primed treatments. e mean
maximum germination rate was observed in Gulistan and CBD Pink Kush for hydro-primed seeds. e mean
minimum germination rate was recorded in KNO3 and un-primed seeds for Fedora-17 and Finola. e CBD
Pink Kush showed a minimum germination rate when seeds were treated with Moringa extract (Fig.6B).
Root length
Overall, the maximum root length was recorded in seedlings treated with Indole Butyric Acid (IBA)
(5.13 ± 0.45cm), followed by hydro-priming (3.90 ± 0.45cm) and potassium nitrate (KNO3). e minimum root
length was observed in seedlings from unprimed seeds (Fig.7). Among the cultivars, Gulistan demonstrated the
best performance across all treatments, followed by CBD Pink Kush, Fedora-17, and Finola. e maximum root
elongation (5.13 ± 0.45cm) in Gulistan may be attributed to its distinct genetic characteristics compared to the
exotic cultivars. Fedora-17 achieved a maximum root length of 3.90 ± 0.45cm under hydro-priming treatment,
while CBD Pink Kush exhibited a maximum root length of 3.47 ± 0.84cm in both hydro-priming and KNO3
treatments (Table2). ese results highlight the signicant inuence of seed priming techniques and cultivar-
specic genetic traits on root development.
Fig. 2. Germination experiment conducted in the lab; (A) Seeds in petri plates, (B) Germination on rst
day, (C) Germination on third day, (D) Germination of Fedora-17 with un-primed seeds, (E) Germination
of Fedora-17 with IBA, (F) Germination of Fedora-17 with Moringa, (G) Germination of Fedora-17 hydro-
primed, (H) Germination of Fedora-17 with KNO3, (I) Length of seedlings on 8th day.
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Shoot length
ere was a varied response in shoot length among the hemp cultivars across dierent growing media.
Overall, hydro-primed seeds exhibited the highest shoot length response in all tested hemp cultivars (Fig.8).
e application of Indole Butyric Acid (IBA) further enhanced the shoot length of all the cultivars studied.
e maximum shoot length was recorded in CBD Pink Kush (0.90 ± 0.08cm), followed by Finola (0.83cm)
and Fedora-17 (0.80cm) (Table2). e signicant improvement in shoot length across all tested cultivars
emphasizes the critical role of IBA in promoting plant growth and development. is nding highlights IBA’s
universal applicability as a growth regulator, underlining its eectiveness in optimizing plant performance and
maximizing yield potential in hemp cultivation.
Seedling Vigor Index
Overall, the maximum seedling vigor index was recorded when seeds were supplemented with 1000 ppm of IBA
treatment, followed by un-primed, hydro-primed, and KNO3 (Fig.9).
CBD Pink Kush showed a maximum seedling vigor index (303) with IBA, followed by un-primed (248.33),
KNO3 (231.3), hydro-primed (218.3), and a minimum of 32 with Moringa. Finola showed a maximum
seedling vigor index of 116.67 in hydro-primed, followed by IBA (28.67), un-primed (18.33), KNO3 (13.33),
and a minimum of 10 with Moringa. Gulistan showed a maximum seedling vigor index of 389.3 in un-primed
seeds, followed by IBA (313.3), KNO3 (238), hydro-primed (202.67), and a minimum of 177.67 in Moringa.
e maximum seedling vigor index of Gulistan in un-primed treatments reects its resilience and natural
compatibility with the native ecosystem (Table2).
Fig. 3. Correlation of hemp cultivars in seed germination: (A): Germination count (partial); (B): Cumulative
germination count; (C): Cumulative seed germination percentage.
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Eect of growing medium on germination potential
For Fedora-17, the germination dynamics varied signicantly across growing media. In hydroponics with liquid
fertilizer, the rst germination was observed aer 2 days, and maximum germination was reached in 5 days at
56%. In cocopeat, germination began aer 3 days, with maximum germination achieved by 7 days at 49.21%.
In soil, germination was slower, with the rst germination occurring aer 5 days and maximum germination
reached in 22 days at 45%.
For CBD Pink Kush, hydroponics demonstrated the fastest response, with the rst germination appearing
aer 1day and maximum germination attained in 6 days at 57.34%. In cocopeat, germination began aer 3 days,
and maximum germination was recorded by 6 days at an impressive 86.23%. In soil, germination was delayed,
starting aer 6 days, and maximum germination was observed in 24 days at 70% (Table3).
ere was varying germination performance in Finola across dierent growth media. In hydroponics
with liquid fertilizer, the rst germination was observed aer 2 days, with maximum germination achieved
by 7 days at 30%. In cocopeat, the rst germination was noted aer 3 days, reaching maximum germination
in 7 days at 67.81%. In soil, germination was slower, beginning aer 8 days, and maximum germination was
attained in 26 days at 42%. For Gulistan, germination showed a more rapid response in hydroponics, where
the rst germination occurred aer 1day, with maximum germination attained by 4 days at 54%. In cocopeat,
germination started aer 3 days, with maximum germination reached in 6 days at 77.07%. In soil, germination
was delayed, rst occurring aer 8 days, and maximum germination was recorded in 28 days at 29.16% (Table3).
Fig. 4. Correlation of hemp seed germination in priming methods; (A): Germination count (partial); (B):
Cumulative germination count; (C): Cumulative seed germination percentage. (Legends: ECV-01 = Fedora-17;
ECV-02 = CBD Pink Kush; ECV-03 = Finola; ECV-04 = Gulistan).
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Eect of IBA treatment on seedling growth uniformity
Seed germination rates were notably improved, and seedling growth was uniform and vigorous across all tested
hemp cultivars when seeds were treated with 1000 ppm of Indole Butyric Acid (IBA). All germination parameters,
including emergence rate, mean germination time, mean germination rate, root length, shoot length, and the
seedling vigor index, showed signicant enhancements under IBA treatment. IBA demonstrated a profound
inuence on the germination dynamics of hemp cultivars. is growth regulator not only enhanced germination
rates and root and shoot development but also supported other critical growth stages, such as owering and
fruit set, in various plant species. Its versatility and eectiveness in stimulating multiple aspects of plant growth
establish IBA as a pivotal tool in agricultural practices and horticultural industries. ese results emphasize IBA’s
potential as a standard priming agent for improving hemp seed germination and seedling vigor, particularly in
industrial and medicinal hemp cultivation.
Fig. 5. Post-hoc testing of dierent hemp cultivars in priming methods: (A) seed germination percentage; (B)
Post-hoc testing results for hemp seed germination percentage across the considered four cultivars and ve
seed priming treatments.
Cultivars Treatments Root length (cm) Shoot length (cm) Germination % SVI
Fedora-17 T02.90 ± 0.22 0.40 ± 0.08 70 ± 8.16 230.33 ± 22.90
Fedora-17 T1 3.07 ± 0.46 0.70 ± 0.08 76.67 ± 9.43 287.67 ± 36.35
Fedora-17 T2 1.57 ± 0.90 0.50 ± 0.08 36.67 ± 9.43 74.67 ± 29.47
Fedora-17 T3 3.90 ± 0.45 0.63 ± 0.12 46.67 ± 9.43 209.33 ± 32.88
Fedora-17 T4 1.77 ± 0.76 0.80 ± 0.08 50.00 ± 0.00 128.33 ± 33.99
CBD Pink Kush T02.53 ± 0.62 0.70 ± 0.14 76.67 ± 4.71 248.33 ± 50.81
CBD Pink Kush T1 2.83 ± 0.85 0.87 ± 0.12 83.33 ± 12.47 303.00 ± 61.58
CBD Pink Kush T2 1.03 ± 0.58 0.33 ± 0.05 20.00 ± 8.16 32.00 ± 23.25
CBD Pink Kush T3 3.47 ± 0.84 0.90 ± 0.08 50.00 ± 0.00 218.33 ± 44.78
CBD Pink Kush T4 3.47 ± 0.96 0.70 ± 0.08 56.67 ± 4.71 231.33 ± 36.53
Finola T00.60 ± 0.42 0.60 ± 0.24 30.00 ± 8.16 18.33 ± 11.15
Finola T1 0.67 ± 0.66 0.17 ± 0.14 30.00 ± 8.16 28.67 ± 33.48
Finola T2 0.13 ± 0.09 0.10 ± 0.00 10.00 ± 8.16 2.00 ± 1.63
Finola T3 3.20 ± 0.57 0.00 ± 0.17 30.00 ± 8.16 116.67 ± 18.06
Finola T4 0.13 ± 0.05 0.83 ± 0.00 13.33 ± 9.43 2.00 ± 1.63
Gulistan T04.43 ± 0.37 0.43 ± 0.05 80.00 ± 0.00 389.3 ± 32.88
Gulistan T1 5.13 ± 0.45 0.60 ± 0.16 53.33 ± 12.47 313.3 ± 106.01
Gulistan T2 4.40 ± 0.43 0.40 ± 0.14 36.67 ± 4.71 177.67 ± 38.82
Gulistan T3 3.80 ± 0.51 0.50 ± 0.14 46.67 ± 4.71 202.67 ± 45.76
Gulistan T4 3.93 ± 0.26 0.50 ± 0.08 53.33 ± 4.71 238.00 ± 39.81
Tab le 2. Seedling Vigor Index of tested hemp cultivars with dierent treatments. T0 = Un-Primed; T1 = IBA;
T2 = Moringa; T3 = Hydro-priming; T4 = KNO3.
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Discussion
is study comprehensively evaluated various medicinal hemp cultivars, examining the eects of selected seed
priming techniques and growth media on seed germination and seedling vigor. Among the treatments, the
maximum germination rates were observed when seeds were primed with 1000 ppm of Indole Butyric Acid
(IBA), followed closely by hydro-priming. Conversely, the minimum germination rates were recorded with
Moringa leaf extract as a priming agent (Fig.3). Similar results were reported by Ghodrat, et al.45 showing that
priming with Indole Butyric Acid (IBA) maximized the seed germination and better establishment of root by
shaping necessary seed physiology. Our results indicated that IBA treatment promoted germination by increase
in counts on the third and h days (Fig.5). is eect may be attributed to inuence on hormonal balance and
cell elongation46. KNO3 treatment also showed a positive inuence on hemp seed germination (Fig.3). ese
ndings are aligned with previous studies reporting the positive eects of IBA and KNO3, application on the
seed germination. Such applications enhance the germination process by providing essential nutrients or acting
as growth regulators47. reported that SA and CaCl2 primed of wheat seeds with signicantly increased rate of
germination; while, our results indicated that hydro priming accelerated the rate of germination. ese ndings
Fig. 7. Root length of considered hemp cultivars in response to dierent seed priming treatments.
Fig. 6. Post-hoc testing of dierent hemp cultivars in priming treatments: (A) mean germination time; (B)
mean germination rate.
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Cultivar Growth medium Days taken for rst germination Days taken for nal germination Germination percentage
Fedora-17
In vitro 2 days 5 days 56%
Cocopeat 3 days 7 days 49.21%
Soil 5 days 22 days 45%
CBD Pink Kush
In vitro 1day 6 days 57.34%
Cocopeat 3 days 6 days 86.23%
Soil 6 days 24 days 70%
Finola
In vitro 2 days 7 days 30%
Cocopeat 3 days 7 days 67.81%
Soil 8 days 26 days 42%
Gulistan
In vitro 1day 4 days 54%
Cocopeat 3 days 6 days 77.07%
Soil 8 days 28 days 29.16%
Tab le 3. Comparative germination of selected hemp cultivars under dierent growth media.
Fig. 9. Seedling Vigor Index (SVI) of considered hemp cultivars in response to dierent priming media.
Fig. 8. Shoot length of tested hemp cultivars in response to considered seed priming media.
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might contribute valuable insights into optimizing medicinal hemp seed germination processes, particularly in
the context of agricultural practices.
e optimum germination of Finola cultivar was recorded in hydro-primed and IBA treatments (80% each).
Contrastingly, an experiment conducted by Pawar, et al.24 showed higher concentration of IBA compared to our
resulted in detrimental eects on germination, seedlings sprouting, and the number of leaves per sprouting. e
germination results demonstrated the ecacy of IBA treatment in enhancing germination percentages for CBD
Pink Kush and Fedora-17. For CBD Pink Kush, the application of IBA resulted in germination percentage of
(83.33) followed by Fedora-17 with germination percentage of 76.67% (Table2). ese results are aligned with
existing literature highlighting the eectiveness of Gibberellic acid and IBA in promoting seed germination48.
Our results are similar to the ndings of Damalas, et al.49 experimented on Faba bean. e hydro-primed seeds
showed a 16.2% higher speed of germination compared to the control. Contrary to the results of Amooaghaie
and Nikzad27 priming with KNO3 media prolonged the germination in all cultivars. Overall, Gulistan exhibited
the fastest germination; whereas, there was a slight dierence in germination rate in Fedora-17 and CBD
Pink Kush. ere was the slowest germination rate in Finola. ese ndings are align with the notion that the
eectiveness of priming agents can vary, emphasizing the importance of selecting appropriate treatments based
on desired outcomes50.
All four cultivars expressed a mean maximum germination rate with hydro-primed treatments. e same
results were reported by Damalas, et al.49 and Adhikari, et al.51. e mean minimum germination rate was
recorded with KNO3 and un-primed seeds for Fedora-17 and Finola. e CBD Pink Kush showed a minimum
germination rate when seeds were treated with Moringa extract. Our results are align with the ndings of
Phiri52 who reported the inuence of moringa leaf extract on germination of major cereal crops. Specically, the
germination percentage of rice was reduced by 7%. e negative eect of Moringa leaf extract on the germination
of hemp seeds could be attributed to several factors such as it contains bioactive compounds like allelochemicals,
which may exhibit allelopathic eects on hemp seeds. ese compounds may inhibit seed germination and
seedling growth by interfering with essential physiological processes53.
e seedling vigor index is a quantitative characteristic that determines the overall performance of seeds
in terms of post-storage performance, emerging ability in unfavorable conditions, growth of seedlings, and
rate and uniformity of germination54. CBD Pink Kush showed a maximum seedling vigor index (303) with
IBA, followed by un-primed (248.33), KNO3 (231), hydro-primed (218.3), and a minimum of 32 with Moringa.
Dhoran and Gudadhe55 reported the same trend for Asparagus sprengeri and the seedling vigor index was found
to be a maximum of 850 when treated with GA3. e results of Chormule, et al.56 also revealed maximum
seedling vigor index i.e. root length and shoot length when seeds of scallion were treated with IBA. Gulistan also
showed a maximum seedling vigor index of 389.3 in un-primed seeds followed by IBA (313.3), KNO3 (238),
hydro-primed (202.67), and a minimum of 177.67 in Moringa (Table2; Fig.9). Khan, et al.57 also reported that
Moringa leaf extract exhibited poor germination in all tested cultivars. e observed inhibition of hemp seed
germination by Moringa leaf extract suggests a potential disruption of key metabolic pathways vital for this
process. It is conceivable that the extract’s bioactive constituents interfere with essential enzymatic activities
crucial for initiating germination. Understanding the exact mechanisms behind Moringa leaf extract’s inhibition
of hemp seed germination requires further investigation. e same results were reported by45 when they primed
seeds of wheat with IBA. ey observed that all the germination parameters were signicantly improved. e
ndings of Kharnaior and omas48 are also align with our results when they primed the seeds with IBA (conc.
200ppm), the speed of germination was increased.
Overall, the maximum root length of hemp cultivars was found in IBA treatment (5.13 ± 0.45), followed
by hydro-priming (3.90 ± 0.45), KNO3 (3.93 ± 0.26), and un-primed (Fig. 3). Among cultivars, Fedora-17
responded well in all treatments, followed by Finola, CBD Pink Kush, and Gulistan (Fig.9B). e application
of IBA enhanced the shoot length of all tested cultivars and the maximum shoot length was recorded in Finola
(0.83cm), followed by Fedora-17 (0.80cm) and CBD Pink Kush (Fig.8). Similar results were reported by Ghafoor,
et al.58 in carrot seeds treated with IBA (25 ppm), revealed maximum root elongation (2.98cm). Ghafoor, et al.58
reported that the dierent plant species may have dierent requirements and responses under such treatments.
e data demonstrated distinct germination patterns among various hemp cultivars and growth media. In
hydroponics with liquid fertilizer, germination response was faster in case of initial germination compared to
cocopeat and soil. However, cocopeat consistently exhibited relatively rapid and uniform germination across all
cultivars59. CBD Pink Kush showed notably higher germination rates in cocopeat compared to other media. Soil,
on the other hand, consistently showed slower germination rates compared to the other media types. e delayed
germination in soil may be attributed to factors such as nutrient availability, soil compaction, and moisture
content, which may vary and impact seedling emergence60. e choice of growth media signicantly inuences
the germination characteristics of hemp seeds. Cocopeat emerges as a promising medium for germination
due to its consistent performance across dierent cultivars, underscoring its potential for application in hemp
cultivation practices.
Conclusions
e study validated the eects of priming techniques and growth media on the germination potential of selected
four hemp cultivars. Hydro-priming revealed the most eective medium for the germination period; while, IBA
priming resulted in a higher germination rate. Similarly, considering all the hemp cultivars, CBD Pink Kush
exhibited the highest seed germination rate. e highest seedling vigor index was observed in Fedora-17, CBD
Pink Kush, and Gulistan supplemented with IBA. is research suggests recommending 24h of IBA (1000ppm)
priming or hydro priming before sowing, along with cocopeat as a growth medium, as standard practice for
farmers and researchers. ese methods would ensure higher germination rates, improve seedling growth and
development, and ultimately lead to increased crop yields. ese insights are vital for agricultural practitioners
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and researchers to consider both variety-specic traits and priming techniques for enhanced crop establishment
and productivity.
Data availability
All the data is available within the article.
Received: 19 June 2024; Accepted: 10 January 2025
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Acknowledgements
e article is extracted from the PhD Dissertation of the rst author. e authors thankfully acknowledge the
APC support provided by the Researchers Supporting Project Number (RSPD2024R966).
Author contributions
Conceptualization, S.L and R.Q; methodology, S.L and S.S.H.S; soware, A.M.K; validation, R.Q, A.R, Q.H and
N.I; investigation, S.L and S.S.H.S.; data curation, S.L, S.S.H.S, A.M.K and R.Q; Writing—original dra prepa-
ration, S.L and S.S.H.S; writing—review and editing, S.L, S.S.H.S, R.Q, A.R and S.R; visualization, N.K and
M.A.A-M; supervision, R.Q, N.I and Q.H; Formal analysis, A.M, S.F, M.A.A-M and B.H.K; Funding acquisition,
A.M, S.F, M.A.A-M and B.H.K. All authors reviewed the manuscript.
Funding
e authors extend their appreciation to the Researchers Supporting Project number (RSP2024R966) King Saud
University, Riyadh, Saudi Arabia.
Declarations
Ethics
e experiment was conducted in compliance with relevant institutional, national, and international guidelines
and legislation.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to R.Q.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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