Shoot Induction Research Articles

Establishment and application of highly efficient regeneration, genetic transformation and genome editing system for cucurbitacins biosynthesis in Hemsleya chinensis

Background Hemsleya Chinensis is a perennial plant in the Cucurbitaceae family containing antibacterial and anti-inflammatory compounds. The lack of genetic transformation systems makes it difficult to verify the functions of genes controlling important traits and conduct molecular breeding in H. chinensis.ResultsHighly efficient calli were induced on MS medium added 1.5 mg·L− 1 6-benzylaminopurine (6-BA) and 0.02 mg·L− 1 1-naphthylacetic acid (NAA) with high efficiency (> 95%). The frequency of shoot induction was increased to 90% with a plant growth regulator combination of 1.5 mg·L− 1 6-BA and 0.1 mg·L− 1 NAA. Our results also showed that 100% of shoot regeneration was achieved in a shoot regeneration medium. Additionally, more than 92% of kanamycin-resistant plants were confirmed. Furthermore, we achieved 42% genome editing efficiency by applying this transformation method to HcOSC6, a gene that catalyzes the formation of cucurbitadienol. HPLC analysis showed OE-HcOSC6 lines exhibited significantly higher cucurbitadienol levels than the genome-edited lines. Transcriptomic analysis revealed that some downstream genes related to cucurbitadienol biosynthesis, such as HcCYP87D20, HcCYP81Q58, and HcSDR34, were up-regulated in OE lines and down-regulated in mutants.ConclusionsHere, we established a process for regeneration, transformation, and genome editing of H. chinensis using stem segments. This provides valuable insight into the underlying molecular mechanisms of medicinal compound production. By combining high-efficiency tissue culture, transformation, and genome editing systems, we provide a powerful platform that supports functional research on molecular mechanisms of secondary metabolism.

Development of In Vitro Regeneration Protocol for Sweet Pepper (Capsicum annuum L.) using Cotyledon as Explant

Plant tissue culture techniques offer a promising avenue for the efficient propagation of sweet pepper (Capsicum annuum L.), a valuable crop with significant agricultural importance. An effective and repeatable in vitro plant regeneration protocol was developed for the CKN-1 (Lemon yellow) and CKN-8 (Red) genotypes of sweet pepper. Utilizing cotyledonary leaf and cotyledonary node explants, various concentrations and combinations of growth regulators were tested for shoot rejuvenation. For both explants, MS medium supplemented with 8 mg/L BAP, 0.02 mg/L NAA, and 0.5 mg/L IAA was found to be most effective for achieving high rates of response and shoot induction. Notably, cotyledonary node explants outperformed cotyledonary leaf explants, displaying a remarkable 80% response within 11-12 days. Additionally, the supplementation of 1 mg/L IBA with MS medium significantly improved root initiation, with cotyledonary node explants exhibiting the highest responsiveness at 83.77%. Throughout all stages, the genotype CKN-8 (Red) consistently outperformed CKN-1 (Lemon yellow), including in the percentage of regenerated plant establishment. J Bangladesh Agril Univ 22(3): 310–316, 2024

Amorphophallus muelleri Blume Shoot Induction on Different Media Types and Plant Growth Regulator Combinations

Amorphophallus muelleri Blume Shoot Induction on Different Media Types and Plant Growth Regulator Combinations

The Elimination of Viroids through In Vitro Thermotherapy and a Meristem Tip Culture from a New Limonime Hybrid (Citrus x limon var. limon (L.) Burm. f. x Citrus latifolia var. latifolia)

Viruses and viroids pose a significant challenge in citriculture, and their control is crucial for plant health. This study evaluated the effectiveness of in vitro thermotherapy combined with a meristem tip culture for eliminating citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) from a new limonime hybrid (Citrus x limon var. limon x Citrus latifolia var. latifolia). The elimination success was confirmed by RT-PCR assays. The in vitro elimination rate for CEVd during the shoot proliferation stage (43%) was higher than for HSVd (21%). Accordingly, in the subsequent rooting stage, the in vitro elimination rate for CEVd (50%) was higher than for HSVd (33%). Successful CEVd and HSVd eradication at a 100% rate was confirmed in the ex vitro acclimatized plants in the greenhouse. The study also established an efficient micropropagation protocol. The optimal treatment for in vitro shoot induction was 0.5–2 mg L−1 benzyladenine (BA) + 0.5 mg L−1 gibberellic acid (GA3) + 0.25 mg L−1 naphthalene acetic acid (NAA), while for shoot elongation, it was 0.5 mg L−1 BA + 0.5 mg L−1 kinetin (KIN) + 0.5 mg L−1 GA3 + 0.25 mg L−1 NAA. Rooting was best promoted by 1 mg L−1 NAA. This study provides valuable insights for the mass production of viroid-free propagation material in this new lemon x lime hybrid, contributing to the conservation of genetic resources in citrus breeding programs through the combined application of in vitro thermotherapy and an in vitro meristem tip culture, a novel and highlighted achievement reported for the first time in this study.

Efficient Plantlet Regeneration from Branches in Mangifera indica L.

Mango (Mangifera indica L.) is one of the most significant tropical and subtropical fruit species, with high ecological and economic value. However, research on the in vitro culture of mangoes is relatively weak, so establishing an efficient and stable mango plant regeneration system is of great significance. In this study, a preliminary mango regeneration system was established with Mangifera indica L. cv. Keitt from young branches as the starting explants. The results showed that the optimal plant growth regulator (PGR) formula for direct adventitious shoot induction on the branches was 1 mg/L 6-benzylaminopurine (6-BA) + 0.1 mg/L a-naphthaleneacetic acid (NAA), with an adventitious shoot induction rate of 73.63% and an average of 6.76 adventitious shoots. The optimal basal medium for adventitious shoot induction was wood plant medium (WPM), with an adventitious shoot induction rate of 63.87% and an average of 5.21 adventitious shoots. The optimal culture medium for adventitious shoot elongation was WPM + 1 mg/L 6-BA + 0.5 mg/L NAA, with an adventitious shoot elongation rate of 89.33% and an average length of 5.17 cm. The optimal formula for the induction of mango rooting was Douglas fir cotyledon revised medium (DCR) + 3 mg/L indole-3-butyric acid (IBA), with a maximum rooting rate of 66.13% and an average rooting quantity of 6.43. The genetic fidelity of the in vitro-regenerated plants was evaluated using inter-simple sequence repeat (ISSR) molecular markers. There was no difference between the in vitro-regenerated plants and the parent plant. This study provides an efficient and stable propagation system for Mangifera indica L., laying the foundation for its rapid propagation and genetic improvement.

Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture.

Development of a Robust In vitro Regeneration Protocol in Cassava (Manihot esculenta C.) Using Axillary Bud Explants for Cryopreservation

Cassava is an important crop as it is the source of energy in the diet for the most of the tropical countries. Field gene bank is the only viable option for the maintenance of cassava germplasm. Cryopreservation offers the long-term conservation of vegetatively propagates crops without losing its viability through an effective in vitro regeneration system. In vitro regeneration response in YTP-1 and Sree Athulya genotypes of cassava was standardized through optimization of various factors influencing organogenesis. Among the different media tested, highest shoot induction frequencies of 81.4% and 77.8% were recorded in medium supplemented with 0.5 mg/l BAP + 0.2 mg/l NAA (CMSI 2) for YTP 1 and Sree Athulya respectively in the present study. 3 to 5 multiple shoots from a single shoot apex obtained from 70-80% of explants within 50-60 days. Highest regeneration frequencies of 56.6%and 46.6% were recorded in medium supplemented with 1 mg/l IAA (CRM 2) for YTP1 and Sree Athulya. As a result, an easy in vitro regeneration method that is genotype independent was created and may be effectively used to preserve cassava germplasm in the cryogen bank.

An Effective Somatic-Cell Regeneration and Genetic Transformation Method Mediated by Agrobacterium tumefaciens for Portulaca oleracea L.

Purslane (Portulaca oleracea L.) is highly valued for its nutritional, medicinal, and ecological significance. Genetic transformation in plants provides a powerful tool for gene manipulation, allowing for the investigation of important phenotypes and agronomic traits at the genetic level. To develop an effective genetic transformation method for purslane, various organ tissues were used as explants for callus induction and shoot regeneration. Leaf tissue exhibited the highest dedifferentiation and regeneration ability, making it the optimal explant for tissue culture. By culturing on Murashige and Skoog (MS) medium supplemented with varying concentrations of 6-benzyleaminopurine (6-BA) and 1-naphthaleneacetic acid (NAA), somatic cells from leaf explants could be developed into calli, shoots, and roots. The shoot induction results of 27 different purslane accessions elucidated the impact of genotype on somatic-cell regeneration capacity and further confirmed the effectiveness of the culture medium in promoting shoot regeneration. On this basis, a total of 17 transgenic plants were obtained utilizing the genetic transformation method mediated by Agrobacterium. The assessment of GUS staining, hygromycin selection, and polymerase chain reaction (PCR) amplification of the transgenic plants as well as their progeny lines indicated that the method established could effectively introduce foreign DNA into the purslane nucleus genome, and that integration was found to be stably inherited by offspring plants. Overall, the present study demonstrates the feasibility and reliability of the Agrobacterium-mediated genetic transformation method for introducing and integrating foreign DNA into the purslane genome, paving the way for further research and applications in purslane genetic modification.

An effective in vitro propagation of Coelogyne mossiae Rolfe: An endangered endemic orchid of Western Ghats, India

Coelogyne mossiae Rolfe, an unexplored taxon given its red-listed status by IUCN was selected for in vitro propagation studies due to its conservational significance. The mature pods harboring viable seeds were subjected to inoculation on eight different nutrient media, revealing full-strength Murashige and Skoog (MS) media as highly efficient for seed germination (96.65 %) and subsequent stages of seedling development. Protocorm explants are used for micropropagation on MS media supplemented with various plant growth regulators (PGRs). α- Naphthaleneacetic acid (NAA) in the concentration of 0.5 mg/L facilitated optimal shoot induction resulting in an average of 2.39 multiple buds and 2.46 cm shoot length while 1.0 mg/L 6-Benzyl amino purine (BAP) augmented cultures provided 2.32 multiple shoot buds with an average 1.92 cm height. Half macro-MS augmented with 0.5 mg/L BAP promoted pseudobulb elongation (0.73 cm) while 0.5 mg/L NAA supported root formation (8.33 roots with an average length of 1.12 cm). The resultant plants were acclimatized in pots containing coco peat and brick pieces, exhibiting a survival rate of 63.23 %. The anatomical characteristics of the hardened plants provided insight into factors conducive to their adaptability. Also, the 3-month-old hardened plants demonstrated significantly greater total photosynthetic pigment (0.4915±0.00 mg g-1), total carbohydrate content (72.33±1.69 mg SSE/g), and total protein content (40.26±0.53 mg BSAE/g) when compared to in vitro plants. These findings underscore the potential of in vitro propagation techniques in the conservation and sustainable utilization of Coelogyne mossiae.

Optimized In vitro Propagation, Acclimatization and Reintroduction of Endemic Rhododendron inaequale Hutch. from Northeast India

Rhododendron inaequale Hutch., an endemic plant with high economic potential and susceptibility, requires urgent conservation measures. This study aimed to develop an optimized in vitro propagation protocol for this species. In vitro seed germination was conducted using Anderson medium, Woody Plant Medium (WPM), and MS Medium. WPM supplemented with 8 mg L⁻¹ 2iP [N6-(2-Isopentenyl) adenine] yielded the highest multiple shoot induction (3.55 ± 0.14 shoots, 0.77 ± 0.03 cm shoot length). For rooting, WPM with 0.5 mg L⁻¹ IBA [Indole-3-butyric acid] produced the maximum number of roots (3.40 ± 0.43) with a mean root length of 1.84 ± 0.12 cm. Successfully acclimatized plants were reintroduced into natural habitats in various locations in Meghalaya and Nagaland. Additionally, 200 micropropagated plants were distributed for further conservation initiatives. This study provides a crucial protocol for the conservation and reintroduction of R. inaequale, aiding in the preservation of this vulnerable species.

Effect of BA on Callus Induction, Shoot and Root Development from Callus of Nepenthes ampullaria through In Vitro Culture

Nepenthes ampullaria Jack. or pitcher plant is a carnivorous plant in the Nepenthaceae family. They are in danger of extinction because of the less forest area, volatile weather, and pollution. This study aimed to investigate the influence of BA (6-benzyl adenine) on callus induction of Nepenthes sp. through In vitro culture. The experiment was divided into three parts; (1) Callus induction; shoot of a pitcher plant were cultured on solid MS medium (Murashige & Skoog, 1962) supplemented with BA (6-benzyladenine) at 0, 1, 2, and 4 mg/l for 70 days. The experiment design was completely Randomized Design (CRD) (4 treatments X 10 replicates). It was found that MS medium supplemented with 4 mg/l BA proved to be a suitable medium for callus induction (70%). (2) Shoot induction from callus; callus were cultured on MS medium supplemented with 0, 2, and 4 mg/l BA (CRD, 3 treatments X 10 replicates) for 45 days. The highest average number of regenerated shoots (19.7±0.3 shoots) was observed on MS medium without BA. (3) Rooting induction; the young shoots, 1-2 cm in length, were cultured on MS medium containing 0, 1, 3, and 5 mg/l NAA combination with 0 and 3 mg/l IBA for 40 days (CRD, 8 treatments X 10 replicates). The results showed that the MS medium supplemented with 1 mg/l NAA and 3 mg/l IBA was suitable for root induction with the average of 7.4±1.6 roots.

Effects of different growth hormones on Solanum tuberosum L. callus induction and regeneration

This study was carried out to develop a protocol for callus induction, regeneration, shoot, and root induction in two potato cultivars (Riviera and Almonda) that can be further used to develop genetically modified plants. Callus was induced from leaf and internodal explants on Murashige and Skoog's medium (MS) supplemented with different combinations of hormones. The highest percentage of callus induction was obtained from the internodal explants of Riviera cultivar was 100% in Callus Induction Media I (CIM I) supplemented with 2 mg/L Benzyl Adenine (BA), 0.2 mg/L ?-Naphthalene Acetic Acid (NAA), 2 mg/L 2,4-Dichlorophenoxy acetic acid (2,4-D), 0.2 mg/L Kinetin (Kin) and 0.2 mg/L gibberellic acid (GA3). After one-month calli were shifted to Shoot Induction Media I and II (SIM I and SIM II), the highest percentage for shoot induction was 33.3% with the leaf explants of Riviera cultivar on SIM II supplemented with 2 mg/L BA, 0.2 mg/L NAA, 0.5 mg/L Kin and 0.2 mg/L GA3. The root induction percentage was 100% for both cultivars. Regenerated potato plants had normal leaf shapes with uniform morphology. These findings provide valuable insights for developing tissue culture protocols for potato regeneration, which are essential for genetic engineering and potato breeding programs.

Ocimum Tenuiflorum Linne: A Narrative Review

Indonesia's diverse flora offers significant potential for medicinal plant production, yet reliance on anecdotal experiences prompts a deeper examination. This study focuses on Ruku-ruku (Ocimum Tenuiflorum Linne), traditionally a culinary herb in West Sumatra. Despite its local culinary use, Ruku-ruku remains largely unexplored for medicinal properties, emphasizing the importance of phytochemical screening to uncover its therapeutic potential. Ocimum Sanctum, known as Holy Basil or Tulsi, holds sacred status in India and boasts a rich history documented in ancient medical texts. This study aimed to review the evidence for therapeutic efficacy and botanical and chemical aspects. The research methodology, employing a narrative literature review, assesses 40 studies (2015-2023). Results underscore Tulsi's holistic benefits, covering metabolic disorders, cardiovascular and cognitive health, infections, cancer prevention, oral health, wound healing, and weight management. Diverse formulations, from essential oils to capsules and gels, highlight the extensive applications of Ocimum Tenuiflorum Linne. The study explores botanical and chemical aspects, including shoot induction and essential oil extraction. Ocimum Sanctum's adaptogenic and antimicrobial properties are discussed, showcasing its potential under stress conditions and against various infections. In conclusion, this review stresses the vast therapeutic potential of Ocimum Tenuiflorum Linne, urging further research to unlock its full medicinal benefits. Integrating traditional wisdom with modern science positions these plants as valuable subjects for pharmaceutical and medicinal applications, potentially addressing contemporary health challenges, including the COVID-19 pandemic. This research contributes to providing information about Ocimum Tenuiflorum Linne and its benefits for health care as therapeutic properties.

Preserving Nature’s Treasure: A Journey into the In Vitro Conservation and Micropropagation of the Endangered Medicinal Marvel—Podophyllum hexandrum Royle

Podophyllum hexandrum Royle, also known as Podophyllum emodi Wall, holds significant ecological, ornamental, and medicinal values. However, it has become endangered due to overexploitation, prolonged seed dormancy, slow natural regeneration, and climate change. This study developed an efficient in vitro protocol for callogenesis and micropropagation of P. hexandrum to conserve germplasm in in vitro conditions. Callus formation from various plant parts, including the leaf, stem, rhizome, radicle, and cotyledon, was induced using Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The combination of benzyladenine at 1 mg L−1 and 4-dichlorophenoxy acetic acid at 3 mg L−1 was optimal for biomass production, yielding 215.88 ± 0.31 mg, with growth per gram at 8.32 ± 0.32 and a growth rate of 13.62 ± 0.25 mg/day on MS medium. For shoot proliferation, benzyladenine (3.5 mg L−1) and naphthalene acetic acid (0.5 mg L−1) combined with activated charcoal showed the highest shoot induction percentage per explant. For shoot regeneration from calluses, 6-benzylaminopurine (0.5 mg L−1) and thidiazuron (2 mg L−1) were most effective, producing superior shoot length, number of regenerations, and regeneration percentage. Root induction was successful with α-naphthalene acetic acid supplementation (0.5 to 1.5 mg L−1) in MS medium, resulting in the highest number per explant (4.08 ± 0.08), length (5.45 ± 0.15 cm), and rooting rate (87.00 ± 1.66%) of roots in plantlets. Subculturing for callus culture was performed every 28 days for up to four subcultures to prevent nutrient depletion and toxic metabolite accumulation, ensuring tissue health and viability. Continuous subculturing of callus on MS medium maintained healthy P. hexandrum germplasm in vitro. Overall, this micropropagation protocol provides a rapid system for conserving P. hexandrum germplasm.

Clonal micropropagation and ex-situ conservation of Rhycncholaelia digbyana (Lindley) Schltr

Objective: To obtain protocols for clonal micropropagation, crop planning, and in vitro conservation of Rhycholaelia digbyana (Lindley) Schltr. Methodology: The effects of the Kundson C basal medium and benzylaminopurine concentration were evaluated for clonal micropropagation. The treatment with the greatest number of shoots formed per apex was selected for crop planning. Experiments were conducted to determine the effect of basal medium Murashige and Skoog concentration at 2.2 gL-1 and 4.4 gL-1; sorbitol, mannitol, and sucrose at 1, 2, and 3% on slow growth. Results: The best treatment for clonal micropropagation and crop planning was identified as 21.60 gL-1 Knudson C with 8.80 µM benzylaminopurine. This treatment resulted in uniform-sized shoots produced. The multiplication process can yield 10,240 seedlings in 12 months. Slow growth was achieved using Murashige and Skoog basal media at 2.2 gL-1 with 1% mannitol. Implications: More experiments must be conducted to determine the best shoot induction conditions and improve resource efficiency. Conclusions: These findings represent the first report on micropropagation and ex-situ conservation to preserve germplasm for this species as an important resource for the floriculture industry.

Propagasi In Vitro Kaliandra Merah (Calliandra calothyrsus Meisn.) II: Induksi Perakaran Tunas dan Aklimatisasi Planlet

Red calliandra is one of woody plants that has been extensively developed as biofuel. Tissue culture techniques are employed to produce a large and uniform quantity of seedlings in a relatively short period. Previous stages of in vitro initiation and multiplication of shoot from calliandra explants have shown promising results. The shoots that were multiplied by in vitro technique need to be induced for rooting process to produce plantlets. These plantlets are then prepared for the subsequent acclimatization process. This research was conducted in two stages; root induction of shoots using a modification of MS half-strength media (MS ½) supplemented with 1-4 mg/L 1-naphthaleneacetic acid (NAA), and acclimatization of the plantlets into soil media. The results shown that MS ½ medium and media supplemented with 1 mg/L NAA were suitable for root induction, particularly to induce the root length. Furthermore, MS ½ media supplemented with 1-2 mg/L NAA stimulates the increment of plantlet height and leaves numbers. Plantlets which were transferred into soil exhibit robust growth during 3-weeks of acclimatization process, up to 100% plantlets survived during this stage. These results underscore the potential of tissue culture techniques in providing large scale calliandra seedlings production for future biofuel development.

In vitro Propagation of Alocasia baginda 'Silver Dragon’ through Direct and Indirect Organogenesis

Direct and indirect in vitro organogenesis of Alocasia baginda 'Silver Dragon' was investigated using explants of shoot tips, leaf tissue, and petiole segments. For direct shoot induction from shoot tips, MS medium containing 1.5 mg/l BAP was shown to be the most efficient (80%), producing a maximum of 6.60 ± 0.93 shoots/explant with 3.66 ± 0.38 cm in length. Directly induced shoots were multiplied using a 2.5 mg/l BAP enriched medium, where the number and length of shoots/culture were 11.60 ± 1.12 and 9.20 ± 1.01 cm, respectively. Shoots gradually increased in number and length during the 1st three sub-culture cycles in the similar medium combination but declined after 3rd cycle. Following the 3rd sub-culture cycle, there were 14.80 ± 0.58 shoots per culture, with a shoot length of 9.90 ± 0.63 cm. The greatest frequency of callus induction was 97.67% for leaf tissue and 86.67% for petiole segments when they were cultured on the media containing 3.0 mg/l 2,4-D. Impressive results regarding indirect shoot organogenesis (90%) with maximum shoot number/unit callus (16.20 ± 1.62) and shoot length (11.06 ± 0.97 cm) were noted upon transferring the callus onto MS medium containing 3.5 mg/l BAP and 3% sucrose. Within 10-12 days, 100% rooting was observed in half-strength of gelled MS medium containing 2.0 mg/l IBA, with an average of 15.40 ± 1.17 roots/culture. After being acclimatized in a potting mixture containing garden soil, compost, coco peat, and moss in a 2:1:1:1 ratio, A. baginda 'Silver Dragon' exhibited a 100% survival rate. Established plantlets seem morphologically similar to mother plants and possess fully developed root and shoot systems, along with an adequate leaf number per plant. Plant Tissue Cult. & Biotech. 34(1): 35-47, 2024 (June)

Applications of combinations and concentrations of different phytohormones for the induction of callus, shoot, and root of cannabis sativaunder tissue culture technique

Cannabis sativa is a significant crop of the family (cannabaceae).Interest has been resurgent in the usage of the cannabis plant for medical purposes, industrial fibre, seed oil, food, and recreation.Auxin and cytokinins act antagonistically with each other in which cytokinins promote shoot branching and inhibit root branching, while auxin inhibits shoot branching and promotes root branching. Young cannabis leaves and petioles were used and inoculated in the MS medium with various combinations. Murashige and Skoog (MS) medium for explant were developed in additions of 25µl-450µl 2, 4 D, 25µl-450µl Kinetin, and 50µl-450µl BAP for callus induction. Shoot induction was developed on 2-3ml/l BAP-supplemented MS media from the callus. Root induction was developed from the shoot on MS5ml/2 litters (indole-3-acetic acid) IAA + 8ml/2 litters (indole-3-butyric acid)IBA-supplemented media.Seeds of cannabis were inoculated in the MS medium. After one week, seed germination was observed in the seeds of the DG Khan variety of Cannabis. MS media found better and more abundant Callus induction with 450µl 2, 4D +450µl Kinetin+450µlBAP from cannabis sativa leaves. 3ml/l BAP observed the high growth of the shoot. Better growth of roots was observed by 5ml/2 litters (indole-3-acetic acid) IAA + 8ml/2 litters (indole-3-butyric acid) IBA. In the present study, seed, callus, shoot, root, and meristem growth were observed using the combination and concentration of different growth regulators. By using different phytohormones, the growth rate and productivity of Cannabiswere significantly increased, which can be valuable for medical purposes.

ESTABLISHMENT OF AN EFFICIENT MICROPROPAGATION PROTOCOLS FOR THREE KENAF (HIBISCUS CANNABINUS L.) CULTIVARS

This study was aimed to establish an efficient micropropagation protocol for three popular kenaf (Hibiscus cannabinus L.) cultivars (V36, HF992 and KB6) using different types of plant growth regulators (PGR’s) on Murashige and Skoog medium (MS). The shoot tip, cotyledon and hypocotyl isolated from 2 weeks old seedlings were used as explants. MS medium fortified with different types and concentrations of cytokinins and auxin were evaluated for establishment callus formation, shoot multiplication and rooting. Among different PGR’s used, 5.0 mg/l 2iP + 0.1 mg/l NAA was the best concentration for direct shoot regeneration from cotyledon with shooting percentage of 55% in HF992 cultivar. In multiple shoot induction experiment, 1.0 mg/l TDZ + 0.1 mg/l NAA mg/l concentration has induced the highest (10.4) shoots/explant. Moreover, indirect shoot regeneration was established from callus which was induced from cotyledon, maximum callus induction percentage (88.75%) was recorded on 0.5 mg/l TDZ and 1.0 mg/l NAA in V36 variety. While, for shoot elongation and rooting, MS media half strength supplemented with 1.0 mg/l kinetin + 1.0 mg/l IBA was found to be an optimum concentration.

Establishment of an <i>In Vitro</i> Propagation, Transformation, and Gene Editing System in Tomato (<i>Solanum lycopersicum</i>)

Tomato (<em>Solanum lycopersicum</em>), a family of Solanaceae, is an annual crop grown in fields and greenhouses. Tomato is the fourth-largest agricultural crop in the world, with a market value of more than $50 billion. Numerous attempts were conducted on the tomato for further improvement via <em>in vitro</em> regeneration and transformation. This study aims to establish <em>in vitro</em> multiplication, transformation, and gene editing systems in tomatoes by using cotyledon explants. Cotyledon explants were placed on the MS medium supported with 6-benzyl adenine (BA) and kinetin (Kin) at four different concentrations for shoot induction. The shoot induction from cotyledon explants significantly increased, and the regeneration rate was 45% after using BA at 8.8 µM compared to Kin. For root induction, indole-3-butyric acid (IBA) at 4.8 µM concentration revealed the highest number of explants producing root (55%). Three <em>Agrobacterium tumefaciens</em> strains (EHA105, GV3101, and LBA4404) were used to establish and develop a tomato transformation method. Each <em>Agrobacterium</em> strain harbors the plasmid pCAMBIA2301, including the <em>nptII </em>marker. <em>Agrobacterium</em> strain LBA4404 showed the lowest number of dead explants, besides increasing the number of explant-produced shoots (30%) and the number of shoots per transformed explants compared to GV3101 and EHA105 strains. Our results revealed that the success of tomato transformation depends on selecting a suitable <em>A.</em> <em>tumefaciens</em> strain. Finally, the tomato gene editing system was successfully established by transferring the pCAMBIA1300-pCas9-sgRNA-U3 binary vector into the cotyledon using the selective <em>Agrobacterium</em> strain LBA4404, and the transformation effectivity was confirmed by PCR analysis.