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Stages of Callus induction and regeneration: (A) Callus induction; (B) Matured callus; (C) Formation of in vitro shoot; (D) Multiple shooting; (E) Profuse in vitro rooting; (F) Plantlet regeneration from callus. Stages of somatic embryogenesis and plantlet formation: (G) Somatic embryo formation; (H) In vitro shooting and rooting from embryo; (I) Fifteen days old plantlet; (J) Plantlet after 1 month; (K) Acclimatization of plant in clay pot containing mixture of soil and sand; (L) Transfer and hardening of in vitro grown plant.
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Canna indica Linn. (Cannaceae) is used both as medicine and food. Traditionally, various parts of C. indica are exploited to treat blood pressure, dropsy, fever, inflammatory diseases etc. However, to date there is no reliable micropropagation protocol for C. indica. We present here a regeneration technique C. indica with banana micropropagation me...
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Context 1
... like structure of the in vitro generated leaves were observed after 15-20 days of culture (Figure 1). Out of various basal media, banana micropropagation media (BM) was found to be the most suitable one. ...
Context 2
... BM with BAP and NAA were proved to be the best for callus induction in Canna. Fully developed callus was noticed after 10-15 days of callus induction (Figure 1). ...
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... auxin (NAA) actually hinders the somatic embryo formation in Canna. Somatic embryos were formed after 10-15 days of transfer of callus into BAP assisted medium (Figure 1). On an average 4-5 somatic embryos were produced per culture vessel. ...
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... in vitro propagated plantlets with well developed shoot and root system were successfully transferred to clay pots containing autoclaved mixture of soil and sand. They were maintained for about 2 months under plastic covers in order to avoid their desiccation (Figure 1). The survival rate of in vitro generated plantlets was as high as 80%. ...
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RAPD and ISSR markers were used to assess the genetic integrity of Indian mustard var. NRCDR-2 plants regenerated from cotyledon explants via direct organogenesis. Of 60 (40 RAPD & 20 ISSR) markers screened, only 27 RAPD and 12 ISSR markers produced a total of 286 (198 RAPD and 88 ISSR) clear, distinct and reproducible amplicons. All the RAPD marke...
Citations
... C. indica significantly reduces BT, CT and abdominal capillary permeability (Al.-Sniff 2015, Woradulayapinij et al. 2005). The hepatoprotective effect of a methanolic extract from the aerial parts of the C. indica L. plant on carbon tetrachloride-induced hepatotoxicity was assessed (Mishra et al. 2015a). ...
... Then artificial culture through tissue meristematic, embryo culture and led to obtaining embryos from seed culture on a completely defined medium. This was followed by establishing the phenomenon of premature germination, thus providing one of the earliest in vitro culture methods (Mishra et al. 2015a(Mishra et al. , 2015b. In vitro germination takes about 18 days to produce a new independent plant. ...
... After a few weeks, the plants are ready to be transferred to soil or other growing medium for further growth and development. In vitro propagation of C. indica is a process that requires high precision and meticulousness, but leads to more healthy and genetically compatible plants in a shorter time (Mishra et al. 2015a, Singh et al. 2019. ...
Ornamental, flowering plants are known all over the world and are a source of nutrients, bioactive molecules and beneficial pharmacological effects. Flowers or whole inflorescences and their bioactive ingredients are becoming more and more popular in the preparation of functional foods, dietary supplements, medicines and industrial products. This chapter summarizes the importance and uses of Canna indica in the horticulture and floriculture industry in recent years. The active components of C. indica are discussed, including: flavonoids, terpenoids, phenylpropanoids, alkaloids, organic acids and others. Antioxidant, anti-inflammatory, anticancer, antiviral and hypoglycemic effects as well as their multifaceted effects have been presented. Due to the richness of nutrients, bioactive ingredients and their wide sources, flowering plants are widely used in research on food, beverages, cosmetics and drugs. C. indica plants also play an important role in the pharmaceutical, food and other industries. A comprehensive overview of the propagation of Canna species is presented, with particular emphasis on C. indica. Genetic and molecular progress in the breeding of this species is discussed.
... Currently, the propagation of the canna lily is mainly by splitting the rhizome, and breeding new canna cultivars rely on traditional breeding methods, which have brought many disadvantages. For example, the reproduction coefficient of conventional methods is extremely low, and long-term clonal propagation is prone to virus accumulation and variety degradation (Mishra et al. 2015). These traditional propagation methods cannot meet the commercial demand in the garden. ...
... The callus was cultured in 2 mg L −1 6-BA medium for 10 to 15 days to form somatic embryos. Each culture flask produced an average of 4-5 individual cell embryos (Mishra et al. 2015). This is the only report about the canna regeneration by somatic embryogenesis, but the induction rate was lower than in the other plants. ...
... Embryogenic callus with uniform growth were cut into 0.5 cm × 0.5 cm in size, inoculated on somatic cell embryo induction medium. Based on previous studies (Mishra et al. 2015), the specific settings were designed and tested at 6-BA (0.0, 1.0, 1.5, 2.0, 2.5, 3 mg L −1 ) and TDZ (0.0, 0.5, 1, 1.5, 2, 2.5 mg L −1 ), which was showed in Table 2. Every treatment was repeated 3 times and 10 bottles per repeat. After culture for 30 days, the induction rate of somatic embryos was calculated according to the following formula. ...
Somatic embryogenesis is a unique method of in vitro regeneration, which can be used in plant reproduction, germplasm conservation, and molecular-assisted breeding. The results showed that the optimum medium for embryogenic callus induction was MS + 6 mg L⁻¹ 6-BA + 1.5 mg L⁻¹ TDZ + 0.5 mg L⁻¹ NAA + 30 g L⁻¹ sucrose + 7 g L⁻¹ agar, and the induction rate was 47.45%. The best somatic differentiation medium was MS + 2 mg L⁻¹6-BA + 1.5 mg L⁻¹ TDZ + 30 g L⁻¹ sucrose + 7 g L⁻¹ agar, and the induction rate of somatic embryos was 54.45%. The optimum medium for embryoid proliferation was MS + 6 mg L⁻¹ 6-BA + 1 mg L⁻¹ NAA + 0.2 mg L⁻¹ TDZ, and the proliferation rate and the multiplication coefficient reached 46.33% and 7.83, respectively. The mature somatic embryos were put into MS, B5, and 1/2MS medium for seedling culture. In MS medium, true leaves grew, complete plants were obtained, and the seedling rate was 88.00%. At the same time, the survival rate of transplanting seedlings in the mixed matrix (peat: organic fertilizer: soil = 1:1:1) was as high as 98%. Cytological observation showed that the somatic embryos underwent globular, heart-shaped, torpedo, and cotyledon stages. This study established a regeneration system of C. × generalis with excellent somatic embryos, and provided basic technical support for the large-scale commercial propagation and germplasm resources protection. It will lay a foundation for further research on gene function and breeding new varieties and ideal research materials for the study of somatic embryogenesis mechanism and genetic transformation of C. × generalis.
... Currently, the propagation of the canna lily is mainly by splitting the rhizome, and breeding new canna cultivars rely on traditional breeding methods, which have brought many disadvantages. For example, the reproduction coe cient of conventional methods is extremely low, and long-term clonal propagation is prone to virus accumulation and variety degradation (Mishra et al. 2015). These traditional propagation methods cannot meet the commercial demand in the garden. ...
... The callus was cultured in 2 mg·L − 1 6-BA medium for 10 to 15 days to form somatic embryos. Each culture ask produced an average of 4-5 individual cell embryos (Mishra et al. 2015). This is the only report about the canna regeneration by somatic embryogenesis; the induction rate was lower than in the other plants. ...
... Table 2). On average, 10-15 somatic embryos produced per 0.5 g embryogenic callus were in good condition, showing white at the early stage and then light green or dark green (Fig. 2), and 4-5 embryos were found to be higher than previous research results (Mishra et al. 2015). Morphological and histological studies were still crucial in the induction of plant embryogenic callus and somatic embryos (Islam et al. 2013). ...
Somatic embryogenesis is a unique method of in vitro regeneration, which can be used in plant reproduction, germplasm conservation, and molecular-assisted breeding. The results showed that the optimum medium for embryogenic callus induction was MS+6 mg L ⁻¹ 6-BA+1.5 mg L ⁻¹ TDZ+0.5 mg·L ⁻¹ NAA+30 g·L ⁻¹ sucrose +7 g·L ⁻¹ agar, and the induction rate was 47.45%. The best somatic differentiation medium was MS+2 mg·L ⁻¹ 6-BA+1.5 mg·L ⁻¹ TDZ+30g·L ⁻¹ sucrose +7g·L ⁻¹ agar, and the induction rate of somatic embryos was 54.45%. The optimum medium for embryoid proliferation was MS +6mg·L ⁻¹ 6-BA + 1 mg·L ⁻¹ NAA +0.2mg·L ⁻¹ TDZ, and the proliferation rate and the multiplication coefficient reached 46.33% and 7.83, respectively. The mature somatic embryos were put into MS, B5, and 1/2MS medium for seedling culture. T In MS medium, true leaves grew, complete plants were obtained, and the seedling rate was 88.00%. At the same time, the survival rate of transplanting seedlings in the mixed nutrient soil with the ratio of original soil (peat: organic fertilizer: soil) =1:1:1 was as high as 98%. Cytological observation showed that the somatic embryos underwent globular, heart-shaped, torpedo, and cotyledon stages. This study established a tissue culture and regeneration system of C. × generalis with excellent somatic embryos, and provide basic technical support for the large-scale commercial propagation and germplasm resources protection. It will lay a foundation for further research on gene function and breeding new varieties and ideal research materials for the study of somatic embryogenesis mechanism and genetic transformation of C. × generalis.
... In the process of tissue culture, the basal medium is the main source of the nutrients required by the explants, and the appropriate basal medium type is essential for the rapid and healthy growth and development of explants from different plant species [29][30][31]. Different parts of the same genotype can have different growth responses to a certain basal medium [32][33][34]. Therefore, the screening of the basal medium is beneficial to the smooth progress of the tissue culture. ...
Rhododendron decorum is a woody species with high ornamental and medical value. Herein, we introduce a novel in vitro regeneration method for R. decorum. We used flower buds to develop an efficient and rapid plant regeneration protocol. Sterile flower buds of R. decorum of a 2 cm size were used as explants to study the effects of the culture medium and plant growth regulators on the callus induction and adventitious shoot differentiation, proliferation, and rooting. According to the results, the optimal medium combination for callus induction was WPM + 1 mg/L TDZ + 0.2 mg/L NAA, and its induction rate reached 95.08%. The optimal medium combination for adventitious shoot differentiation from the callus was WPM + 0.5 mg/L TDZ + 0.1 mg/L NAA, and its differentiation rate reached 91.32%. The optimal medium combination for adventitious shoot proliferation was WPM + 2 mg/L ZT + 0.5 mg/L NAA, for which the proliferation rate reached 95.32% and the proliferation coefficient reached 9.45. The optimal medium combination for rooting from adventitious shoots was WPM + 0.1 mg/L NAA + 1 mg/L IBA, and its rooting rate reached 86.90%. The survival rates of the rooted regenerated plantlets exceeded 90% after acclimatization and transplantation. This regeneration system has the advantages of being simple and highly efficient, and it causes little damage to the shoots of the mother plants, laying a foundation for the plantlet propagation, genetic transformation, and new-variety breeding of R. decorum.
... Similar result was obtained from the heterozygosity proportion distribution of the 56 banana samples. Molecular markers ( e.g. , SSR, ISSR and RAPD) have been widely used in crops for genetic relationship analysis [52][53][54] including SNP markers [55][56] that are most widely accepted nowadays in worldwide molecular research. This study suggests that banana accessions, which are true-to-type between field and in vitro lines, can be used for large biomass production of Musa spp. ...
Bananas and plantains (Musa spp.) are used as nutritious foods, and at the same time, are a source of phytoconstituents for the pharmaceutical industry. As biological activities of especially the pulp and peel of Musa spp. have been documented, this study investigated the variation in the secondary metabolite profiles of the leaves from field, in vitro-grown and acclimatized accessions. The genetic fidelity of the diverse accessions was assessed using diversity array technology sequencing. It showed that the in vitro-grown accessions were true-to-type with the field samples. The antioxidant and anticholinesterase activities of the samples from different culture systems (field and in vitro) were evaluated by UV-spectrophotometry and compared to high-performance thin-layer chromatography-effect-directed analysis (HPTLC-EDA). The latter was applied for the first time for effect-directed profiling of the polar and medium polar sample
components via different biochemical and biological assays. Compound zones showed acetyl- /butylrylcholinesterase inhibition (zones 1-4), α-/β-glucosidase inhibition (zones 1 and 2) as well
as antioxidative (zones 1-3) and antimicrobial (zone 4) activities. Structures were preliminary assigned by HPTLC-HRMS. The HPTLC was effective for bioactivity-guided characterization of the bioactive constituents in Musa spp. accessions. Accumulation of useful metabolites, especially compounds with antioxidant and anticholinesterase properties, was higher in samples from in vitro system. This validated the use of plant tissue culturing as an alternative method for large scale production of plant material and supply of bioactive constituents.
... Controlled cultures with no hormone were also prepared. Sub-culturing was done at 2 weeks interval in the same media having the same hormonal composition [15]. BAP was applied at the rate of 1, 2, 3 and 4 mg/l and NAA at the rate of 0.5mg/l to observe various stages of in-vitro callusing and subsequent shoot formation. ...
Clerodendrum thomsoniae commonly known as bleeding heart vine or bag flower which is a good candidate for a new crop for the floriculture industry. In this study, in-vitro callus regeneration of C. thomsoniae through nodal culture has been attempted. Murashige and Skoog’s medium supplemented with 2 mg/l BAP and 0.5 mg/l
... Developing a reliable whole-plant regeneration protocol is essential for the genetic improvement of Canna. Till date, there are five reports on in vitro regeneration from aseptically cultured explants of Canna indica and C. edulis (Kromer, 1979;Kromer and Kukulczanka, 1985;Hosoki and Sasaki, 1991;Sakai and Imai, 2007;Mishra et al., 2015). Earlier investigators have exclusively utilised the apical shoot tip and cultured it on MS medium supplemented with cytokinins notably BA, that was found to be the most efficient for the regeneration of shoots as compared to kinetin, zeatin, and dimethylallylaminopurine (2-iP). ...
Canna being ornamental plants has a significant role in agriculture, medical, economy and food industry. Canna has a limited vase life due to the rapid loss of moisture from its perianth. For improving its market value, cuticularisation of the perianth can be achieved by the expression of a heterologous cutin producing gene, using the tissue culture and transformation protocol developed in this study. Efficient, rapid and direct adventitious shoot regeneration was successfully established in Canna × generalis using recalcitrant rhizome explants. The explants were cultured on MS medium supplemented with 6-benzylaminopurine (6-BA), thidiazuron (TDZ), and kinetin. Among the four genotypes taken for tissue culture, the ‘Trinacria variegata’ was the best responding cultivar. And 2 mg · L ⁻¹ 6-BA or 1.5 mg · L ⁻¹ TDZ along with 0.1 mg · L ⁻¹ IAA was optimum for their regeneration. The highest regeneration was achieved in ‘Trinacria variegata’ (36%) on 6-BA, 33% on TDZ while kinetin failed to evoke any regenerative responses. Regeneration was enhanced by supplementation of 100 mg · L ⁻¹ ascorbic acid (AsA), while, 100 mg · L ⁻¹ of L-cysteine or 100 mg · L ⁻¹ dithiothreitol (DTT), inhibited regeneration. Shoots were observed to develop 3–5 fibrous roots on MS medium supplemented with 0.5 mg · L ⁻¹ indole-3-butyric acid (IBA). The plantlets were transplanted into pots and acclimatised in glasshouse with 100% survival. For transformation of Canna, rhizome explants were co-cultivated for 60 min in Agrobacterium suspension. The explants were washed with 500 mg · L ⁻¹ cefotaxime solution, subjected to 100 mg · L ⁻¹ kanamycin selection followed by excision of the shoots and culturing them on IBA-supplemented media for root development. Transgene integration in the putative transformants was confirmed by PCR assay and copy number by Southern blot hybridisation analysis. © 2018 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)
... Despite its many medicinal values and uses, traditional propagation of C. indica is hindered by its extremely hard seed coat [9] and slow vegetative propagation of rhizome which is also susceptible to viral infection during multiplication [10][11][12][13]. Furthermore, common practice of asexual propagation through rhizome produces genetic stagnancy and genetic variation limitations in Canna [14]. Plant tissue culture provides a solution whereby the method rapidly micropropagates plants of superior qualities and free from microorganisms in a relatively short time and minimal space using few starting materials. ...
... Later, the same author tried to stimulate shoot development or bud formation from meristem tips with little success [16]. Recently, a study managed to obtain complete plantlets from leaves explant of C. indica via indirect regeneration through in vitro callus and somatic embryogenesis [14]. ...
... According to the table, the percentage of shoot formation increased with the increase of BAP up to the optimum concentration of 5.0 mg/L and later gradually decreased. The pattern was in agreement with previous researchers [14,23] which observed a less effective formation of shoots with higher concentrations of BAP. The variants in optimum concentration of BAP for regeneration could be attributed to the types of explants used. ...
... Despite its many medicinal values and uses, traditional propagation of C. indica is hindered by its extremely hard seed coat [9] and slow vegetative propagation of rhizome which is also susceptible to viral infection during multiplication [10][11][12][13]. Furthermore, common practice of asexual propagation through rhizome produces genetic stagnancy and genetic variation limitations in Canna [14]. Plant tissue culture provides a solution whereby the method rapidly micropropagates plants of superior qualities and free from microorganisms in a relatively short time and minimal space using few starting materials. ...
... Later, the same author tried to stimulate shoot development or bud formation from meristem tips with little success [16]. Recently, a study managed to obtain complete plantlets from leaves explant of C. indica via indirect regeneration through in vitro callus and somatic embryogenesis [14]. ...
... According to the table, the percentage of shoot formation increased with the increase of BAP up to the optimum concentration of 5.0 mg/L and later gradually decreased. The pattern was in agreement with previous researchers [14,23] which observed a less effective formation of shoots with higher concentrations of BAP. The variants in optimum concentration of BAP for regeneration could be attributed to the types of explants used. ...
An efficient protocol for micropropagation of Canna indica L., an economically and pharmaceutically important plant, was standardized using rhizome explants, excised from two-month-old aseptic seedlings. Complete plant regeneration was induced on MS medium supplemented with 3.0 mg/L BAP plus 1.5 mg/L NAA, which produced the highest number of shoots (73.3 ± 0.5%) and roots (86.7 ± 0.4%) after 2 weeks. Furthermore, the optimum media for multiple shoots regeneration were recorded on MS enriched with 7.0 mg/L BAP (33.0 ± 0.5%). Plantlets obtained were transplanted to pots after two months and acclimatized in the greenhouse, with 75% survival. In addition, ultrastructural studies showed that rhizomes of in vitro grown specimens were underdeveloped compared to the in vivo specimens, possibly due to the presence of wide spaces. Meanwhile, the leaves of in vivo specimens had more open stomata compared to in vitro specimens, yet their paracytic stomata structures were similar. Hence, there were no abnormalities or major differences between in vitro regenerates and mother plants.
Ornamental flowering plants, exemplified by Canna indica L., have garnered global acclaim not solely for their visual allure but also as reservoirs of nutrients and bioactive compounds with diverse pharmacological effects. This chapter articulates a strategy for preserving and augmenting genetic diversity within C. indica populations. The aim is to thwart genetic stagnation, fortify variety resistance, and contribute to the creation of visually captivating and commercially viable C. indica varieties boasting enhanced bioactive properties. The chapter provides a succinct overview of the significance and applications of C. indica in the horticulture and floriculture industries. It accentuates the active components present in C. indica, encompassing flavonoids, terpenoids, phenylpropanoids, alkaloids, organic acids, and others. Delving into the multifaceted effects of C. indica, including antioxidant, anti-inflammatory, anticancer, antiviral, and hypoglycemic properties, the text explores the broad spectrum of its pharmacological contributions. In the context of flowering plants' rich nutritional and bioactive diversity, pivotal roles have emerged in research pertaining to functional foods, dietary supplements, cosmetics, and pharmaceuticals. C. indica, with its diverse compounds, assumes a critical position in pharmaceutical, food, and various other industries. The chapter delivers a thorough examination of Canna species propagation, placing particular emphasis on C. indica. Furthermore, it elucidates genetic and molecular progressions in the breeding of C. indica, underscoring continuous endeavors aimed at refining and augmenting the potential of this invaluable plant species.
