Development and Standardization of Rapid and Efficient Seed Germination Protocol for Cannabis sativa

Sorokin, Aleksei; Yadav, Narendra Singh; Gaudet, Daniel; Kovalchuk, Igor

doi:10.21769/bioprotoc.3875

Cited by 9 publications

(8 citation statements)

References 6 publications

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“…sativa plants and found that 1% H 2 O 2 can be used as a liquid germination medium for aseptic in vitro germination, consistent with previous findings (Sorokin et al 2021).…”

Section: Discussionsupporting

confidence: 91%

“…Based on these two positive attributes of H 2 O 2 (as a germination inducer and an antimicrobial agent) and results of a Availability of Hydrogen Peroxide Solutions as a Germination Liquid Medium for Contamination-free in vitro Seedling Development of ... previous report by Sorokin et al (2021), different concentrations of H 2 O 2 as a liquid germination medium against four different cultivars of C. sativa plants, in this case Cheungsam, Cherry Blossom, Queen Dream, and Hot Blonde, were tested in this study. As expected, 1% H 2 O 2 significantly enhanced the seed germination rate while also significantly and drastically reducing contamination rates of all tested C. sativa seeds.…”

Section: Introductionmentioning

confidence: 99%

“…Recently, it has been reported that a 1% hydrogen peroxide (H 2 O 2 ) solution can significantly enhance the germination rate of C. sativa seeds (Sorokin et al, 2021). H 2 O 2 is not only a well-established antimicrobial agent (Weston, 2000) but is also a signaling molecule for many physiological, developmental, and stress-tolerance processes of plants (Wojtyla et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

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Availability of Hydrogen Peroxide Solutions as a Germination Liquid Medium for Contamination-free in vitro Seedling Development of Cannabis sativa

Ahsan,

Shin,

Choi

2022

HST

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Endophytic and seed-borne microbial attacks represent major barriers leading to poor regenerationpowered micro-propagation of Cannabis sativa. Thus, a reliable sterile method during germination and in vitro plantlet preparation is important for the further successful micro-propagation of C. sativa plants. In the present study, we aim to develop a quick and efficient procedure for the preparation of pathogen-free plantlets using a hydrogen peroxide (H 2 O 2 ) solution as a liquid germination medium. In this framework, all three phases, i.e., seed sterilization, germination, and early seedling development, were carried out in H 2 O 2 solutions of varying concentrations (0, 1, 3, 5, and 10%). Among the different concentrations tested, the 1% H 2 O 2 solution resulted in the fastest and the most successful germination for all tested genotypes of C. sativa seeds, in this case "Cheungsam," "Cherry Blossom," "Queen Dream," and "Hot Blonde." Higher concentrations of H 2 O 2 (3, 5, and 10%) adversely affected the germination rate of C. sativa seeds compared to 0% or 1% H 2 O 2 . In addition, 5% and 10% H 2 O 2 solutions significantly reduced seedling survival rates on MS media. Importantly, contamination rates of 88.4-96.7% were observed for C. sativa seedlings germinated with the 0% H 2 O 2 solution and transferred to antibiotic-free MS media. However, a contamination rate of less than 4.0% was observed for seedlings prepared using 1% H 2 O 2 as a germination medium. Moreover, the survival rates of seedlings germinated in 1% H 2 O 2 were significantly higher than those of other seedlings. Our results suggest that a 1% H 2 O 2 solution is a useful liquid germination medium for C. sativa seeds to enhance germination and survival rates while decreasing microbial contamination in antibiotic-free tissue culture media.

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“…sativa plants and found that 1% H 2 O 2 can be used as a liquid germination medium for aseptic in vitro germination, consistent with previous findings (Sorokin et al 2021).…”

Section: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Availability of Hydrogen Peroxide Solutions as a Germination Liquid Medium for Contamination-free in vitro Seedling Development of Cannabis sativa

Ahsan,

Shin,

Choi

2022

HST

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“…The combination priming treatment significantly improved germination index which is notable as there is currently no published data on this combination of priming. As previously stated, both the H 2 O 2 and micronutrient priming treatments have been shown to increase germination and various factors in plant growth 10,19,26,30 including in hemp 31 . The results here support the notion that priming hemp seeds with H 2 O 2 and micronutrients may produce a synergistic effect.…”

Section: Discussionmentioning

confidence: 67%

The role of oxidative stress in seed priming to improve germination and vigour

Marks

Cowley

Bianco‐Miotto

et al. 2022

Preprint

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Seed priming improves germination, but responses vary with procedure and plant used, potentially from differential responses in oxidative pathways. This study targeted oxidative pathways in seed priming, using hydrogen peroxide (H2O2), antioxidant-related micronutrients (Zn, Se and Mn), or a combination, to modulate germination and plant growth characteristics of hemp and barley. Hemp tolerated higher H2O2 (1 M vs 0.125 M) and micronutrients (6-fold greater) concentrations than barley, with the combination treatment significantly increasing hemp germination but decreasing barley germination. Seed priming did not improve hemp germination under salt stress (200 mM NaCl), but the micronutrient treatment improved germination of barley under salt stress (100 mM NaCl). Histological staining showed that micronutrient-primed root tips accumulated less O2- in both non-stressed and salt-stressed conditions. We assessed seed priming benefits if grown in soil by measuring plant quality in three-week-old plants potted post-priming, but most quality factors were not significantly improved, except barley where shoot zinc content increased. In summary, seed priming with H2O2 and/or micronutrients increased germination in hemp, but only micronutrients increased barley germination. Future work will continue optimising the priming methodology and further investigate the role of oxidative stress in the observed responses.

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“…Wahby et al [ 23 ] used different parts of 5-day-old in vitro-grown seedlings such as hypocotyls, cotyledons, cotyledonary nodes, and primary leaves for gene transformation, and their report states that the best gene transformation results were obtained from hypocotyl segments. Sorokin et al [ 42 , 43 ] also used cotyledons and true leaves of 4-day-old in vitro-grown hemp seedlings for gene transformation. Deguchi et al [ 2 ] reported a successful gene transformation using male and female flowers, stems, leaves, and root tissues derived from 2-month-old in vitro-grown hemp seedlings.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Parameters Affecting Agrobacterium-Mediated Transient Gene Expression in Industrial Hemp (Cannabis sativa L.)

Mohammad,

Ghogare,

Morton

et al. 2024

Plants

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Industrial hemp Cannabis sativa L. is an economically important crop mostly grown for its fiber, oil, and seeds. Due to its increasing applications in the pharmaceutical industry and a lack of knowledge of gene functions in cannabinoid biosynthesis pathways, developing an efficient transformation platform for the genetic engineering of industrial hemp has become necessary to enable functional genomic and industrial application studies. A critical step in the development of Agrobacterium tumefaciens-mediated transformation in the hemp genus is the establishment of optimal conditions for T-DNA gene delivery into different explants from which whole plantlets can be regenerated. As a first step in the development of a successful Agrobacterium tumefaciens-mediated transformation method for hemp gene editing, the factors influencing the successful T-DNA integration and expression (as measured by transient β-glucuronidase (GUS) and Green Florescent Protein (GFP) expression) were investigated. In this study, the parameters for an agroinfiltration system in hemp, which applies to the stable transformation method, were optimized. In the present study, we tested different explants, such as 1- to 3-week-old leaves, cotyledons, hypocotyls, root segments, nodal parts, and 2- to 3-week-old leaf-derived calli. We observed that the 3-week-old leaves were the best explant for transient gene expression. Fully expanded 2- to 3-week-old leaf explants, in combination with 30 min of immersion time, 60 µM silver nitrate, 0.5 µM calcium chloride, 150 µM natural phenolic compound acetosyringone, and a bacterial density of OD600nm = 0.4 resulted in the highest GUS and GFP expression. The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest, using the latest technologies such as genome editing, and studying gene functions that regulate secondary metabolites in hemp.

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Development and Standardization of Rapid and Efficient Seed Germination Protocol for Cannabis sativa

Cited by 9 publications

References 6 publications

Availability of Hydrogen Peroxide Solutions as a Germination Liquid Medium for Contamination-free in vitro Seedling Development of Cannabis sativa

Availability of Hydrogen Peroxide Solutions as a Germination Liquid Medium for Contamination-free in vitro Seedling Development of Cannabis sativa

The role of oxidative stress in seed priming to improve germination and vigour

Evaluation of Parameters Affecting Agrobacterium-Mediated Transient Gene Expression in Industrial Hemp (Cannabis sativa L.)

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