Development of plant tissue culture method for various genotypes of Cannabis sativa

다국어 초록 (Multilingual Abstract)

GENERAL ABSTRACT
Development of plant tissue culture method for various genotypes
of Cannabis sativa
S.M. AHSAN
Major in Plant Pathology
Department of Plant Medicals, Gyeongkuk National University
Endophytic and seed-borne microbial attacks represent major barriers leading to
poor regeneration-powered 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 (H₂O₂) 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 H₂O₂ solutions
of varying concentrations (0, 1, 3, 5, and 10%). Among the different
concentrations tested, the 1% H₂O₂ solution resulted in the fastest and the most
successful germination for all tested genotypes of C. sativa seeds(derived from
controlled condition), in this case “Cheungsam,” “Cherry Blossom,” “Queen
Dream,” and “Hot Blonde.” Higher concentrations of H₂O₂ (3, 5, and 10%) adversely affected the germination rate of C. sativa seeds compared to 0% or 1%
H₂O₂. In addition, 5% and 10% H₂O₂ 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% H₂O₂ solution and
transferred to antibiotic-free MS media. However, a contamination rate of less
than 4.0% was observed for seedlings prepared using 1% H₂O₂ as a germination
medium. Moreover, the survival rates of seedlings germinated in 1% H₂O₂ were
significantly higher than those of other seedlings. Our results suggest that a 1%
H₂O₂ 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. Among the 4 genotypes Cannabis sativa L.
cv. ‘Cheungsam’ is an industrial hemp plant of Republic of Korea origin,
primarily cultivated for fiber and seed production. In another study, we aimed to
develop a tissue culture process for hemp plants using Cheungsam as a model
plant and examine the secondary metabolites produced from its callus. We also
developed a method to prepare pathogen-free seedlings from field-derived seeds
using hydrogen peroxide (H2O2) solution as a liquid germination medium.
Treating seedlings with removed seed coat in 3% H2O2 significantly reduced the
contamination rate. Callus formation and de novo organogenesis of shoots and
roots from callus were successfully achieved using cotyledon and leaf tissues
prepared from the pathogen-free seedlings. The most effective in vitro regeneration results were obtained using the Murashige and Skoog (MS) medium
supplemented with certain targeted growth regulators. An optimal combination of
0.5 mg/L thidiazuron (TDZ) and 1.0 mg/L 1-naphthalene acetic acid proved
highly effective for callus induction. The addition of 0.5 mg/L TDZ in the MS
medium significantly stimulated shoot proliferation, while robust root
development was best supported by MS medium supplemented with 2.5 mg/L
indole-3-butyric acid for both cotyledon and leaf explants. Finally, gas
chromatography–mass spectrometry (GC–MS) analysis of ethanol extract from
Cheungsam leaf callus revealed the presence of different secondary metabolites,
including 9-octadecenamide, methyl salicylate, dodecane, tetradecane, and phenol,
2,4-bis-(1,1-dimethylethyl). This study provides a comprehensive de novo
regeneration protocol for Cheungsam plants and insight into the secondary
metabolite profiles of its callus. Cannabis sativa is relatively recalcitrant to de
novo shoot regeneration. While different in vitro techniques are instrumental to
the development of the medicinal plant industry, the challenge of inherent
regeneration recalcitrance in some species to in vitro cultivation hampers these
efforts. Here we attempted to replicate our published Cannabis regeneration study
for Cheungsam and expand the original scope of this study by testing it across 3 C.
sativa cultivars (“Cherry Blossom” “Queen Dream” and “Hot Blonde”) to assess
its reproducibility. In our study, leaf and cotyledon explant taken from (1% H2O2
treated in vitro seedling) derived callus was induced in all 3 cultivars in CIM but lower shoot response substantially differed among cultivars, with the most
responsive genotype (Cherry blossom) producing more shoots from cotyledon
derived callus in SIM than the other two cultivars tested. Robust root development
was supported by RIM for shoots obtained from shoot cultures derived from
cotyledon and leaf explants in all the cultivars. The findings of this replication
study raise concerns about the replicability of existing methods. These results
highlight the importance of using multiple genotypes in such studies and
providing detailed methods to facilitate replication.

목차 (Table of Contents)

• 1 Origin and Classification of Cannabis sativa……………… 1
• 2 Manifold Application of Cannabis sativa…………………. 6
• 3 Secondary metabolism in Cannabis sativa and their role in
• human body…………………………………………………. 10
• 4 Plant tissue culture is the gate way of different
• biotechnological application in Cannabis sativa …………… 13
• CHAPTER 1. Availability of Hydrogen Peroxide Solutions as a Germination
• Liquid Medium for Contamination-free in vitro Seedling Development of
• Cannabis sativa
• Title Page
• Abstract……………………………………………………………. 23
• 1 Introduction………………………………………………………. 25
• 2 Materials and Methods……………………………………………. 29
• 2.1 Cannabis sativa Seeds……………………………………………. 29
• 2.2 Seed Germination Assay………………………………………. 29
• 2.3 In vitro Development of Germinated Seedlings…………………. 30
• 2.4 Data Analysis…………………………………………………… 30
• 3 Results……………………………………………………….…… 31
• 4 Discussion……………………………………………………… 38
• CHAPTER 2. De Novo Regeneration of Cannabis sativa cv. Cheungsam and
• Evaluation of Secondary Metabolites of Its Callus
• Title Page
• Abstract……………………………………………………… 42
• 1 Introduction……………………………………………………. 44
• 2 Methods and Materials……………………………………… 50
• 2.1 Optimization of Sterilization Methods for the Generation of
• Pathogen-Free Seedlings……………………………………… 50
• 2.2 Callus, Shoot, and Root Induction from Cotyledon and Leaf
• Explants………………………………………………………. 52
• 2.3 GC–MS Measurement of Secondary Metabolites in Callus
• Cultures………………………………………………………. 54
• 2.4 Data analysis…………………………………………………. 55
• 3.3 Result………………………………………………………… 56
• 3.1 Preparation of Pathogen-Free Seedlings…………………… 56
• 3.2 In vitro Callogenesis from Leaf and Cotyledon Explants…… 60
• 3.3 De Novo Shoot Morphogenesis from Callus…………………. 68
• 3.4 De Novo Root Morphogenesis from Callus-Derived Shoots… 70
• 3.5 Gas Chromatography–Mass Spectrometry (GC–MS) Analysis 71
• 4 Discussion…………………………………………………… 74
• 5 Conclusions…………………………………………………… 82
• CHAPTER 3. Reproducibility trial of protocol developed from Cannabis
• sativa cv. Cheungsam on other 3 genotypes for observing recalcitrance nature
• Title Page
• Abstract……………………………………………………… 84
• 1 Introduction………………………………………………… 86
• 2 Materials and Methods………………………………………. 92
• 2.1 In vitro Development of Germinated Seedlings from 3
• genotypes……………………………………………………… 92
• 2.2 Callus, Shoot, and Root Induction from Cotyledon and Leaf
• Explants of 3 genotypes……………………………………….
• 94
• 2.3 Experimental design and statistical analysis……………… 96
• 3 Results………………………………………………………… 97
• 3.1 In Vitro Callogenesis from Leaf and Cotyledon Explants of
• Cherry blossom, Hot Blonde and Queen Dream…………….
• 97
• 3.2 De Novo Shoot Morphogenesis from leaf and cotyledon
• derived callus of 3 cultivars………………………………….
• 107
• 3.3 De Novo Root Morphogenesis from Callus-Derived Shoots of
• 3 cultivars…………………………………………………….
• 108
• 4 Discussion…………………………………………………… 109
• 5 Conclusion…………………………………………………… 114
• REFERENCES………………………………………… 116
• KOREAN ABSTRACT……………………………………. 136