Tissue Culture Propagation Research Articles

Global characterization of somatic mutations and DNA methylation changes during vegetative propagation in strawberries.

Somatic mutations arise and accumulate during tissue culture and vegetative propagation, potentially affecting various traits in horticultural crops, but their characteristics are still unclear. Here, somatic mutations in regenerated woodland strawberry derived from tissue culture of shoot tips under different conditions and 12 cultivated strawberry individuals are analyzed by whole genome sequencing. The mutation frequency of single nucleotide variants is significantly increased with increased hormone levels or prolonged culture time in the range of 3.3 × 10-8-3.0 × 10-6 mutations per site. CG methylation shows a stable reduction (0.71%-8.03%) in regenerated plants, and hypoCG-DMRs are more heritable after sexual reproduction. A high-quality haplotype-resolved genome is assembled for the strawberry cultivar "Beni hoppe." The 12 "Beni hoppe" individuals randomly selected from different locations show 4731-6005 mutations relative to the reference genome, and the mutation frequency varies among the subgenomes. Our study has systematically characterized the genetic and epigenetic variants in regenerated woodland strawberry plants and different individuals of the same strawberry cultivar, providing an accurate assessment of somatic mutations at the genomic scale and nucleotide resolution in plants.

Targeting Cancers: Uncovering the Potential Roles of Potato Tissue Culture as Anti-Cancer Agents — A Secondary Publication

Plant tissue culture is a technique that enhances the quality and quantity of potatoes. Potatoes are a significant crop and are primarily used in the world. It is a staple food in many countries, where millions of tonnes are produced annually. It is an essential source of many nutrients, such as proteins, carbohydrates, vitamins, and beta-carotene. In addition, potatoes are being used as therapeutic agents against cancer and other human diseases as well. Potatoes are on the third list after wheat and rice. To overcome food shortages and malnutrition, there are two methods used for producing potatoes: the first is sexual, which is seed propagation, and the second is asexual, which is plant tissue culture propagation. Conventional potato breeding is a uniform method, but it is unsafe because there is a risk of pathogen attack. In a laboratory setting, the tissue culture of potatoes produced millions of plants with nutrient-rich medium under controlled environmental conditions that prevent pest attacks. Some environmental stresses, such as salinity and water scarcity, affect potato yield and production; however, applying nanoparticles like organic, inorganic, and silicon dioxide enhances potato quality and combats stress. Biotechnology has proven to be helpful in addressing all these issues. This review discusses the significance of potatoes, their production through the tissue culture technique, and the application of nanoparticles to improve the growth, and impact of potatoes on human health.

Potato (Solanum tuberosum L.) Planlet Acclimatization on Various Auxin Concentrations and Growing Media

The provision of quality potato seeds is a problem faced by farmers. Tissue culture propagation is an effort to increase the availability of quality seeds. The success of tissue culture is influenced by the ability to control environmental conditions, growth regulators, and growing media at the acclimatization stage. The research aims to determine the interaction between the use of auxin concentrations and the planting medium and to obtain the appropriate concentration of auxin and planting medium for potato acclimatization. The research used a split-plot design with 2 factors and 3 replications. The main plot is the auxin concentration consisting of 3 levels; 1 ppm, 2 ppm, and 3 ppm. Subplots are planting media consisting of 3 levels; husk charcoal:vermicompost, cocopeat:vermicompost, and husk charcoal:cocopeat:vermicompost. Data were analyzed using ANOVA and followed by DMRT at a 5% level. The results showed that there was an interaction between the treatment of 3 ppm IAA concentration and the planting medium of husk charcoal:cocopeat:vermicompost on the plant height parameter 4 WAP. The IAA concentration of 3 ppm was better than the concentration of 1 ppm in the parameter number of leaves 8 WAP. The growing media of husk charcoal:cocopeat:vermicompost was better than the growing medium of charcoal husk:vermicompost on the parameters of plant height 8 WAP, number of leaves 8 WAP, leaf length and leaf width 8 WAP.

An Efficient Method for the Propagation of Bougainvillea glabra ‘New River’ (Nyctaginaceae) from In Vitro Stem Segments

Bougainvillea, an evergreen climbing shrub of the Nyctaginaceae family, holds significant ornamental, economic, and medicinal value. Bougainvillea glabra ‘New River’ is widely used in landscapes due to its strong adaptability to the environment, abundance of flowers, and frequent flowering. Traditionally, Bougainvillea glabra ‘New River’ cultivation has relied on methods such as cuttings or grafting, with limited research on in vitro tissue culture propagation. This study aimed to optimize the tissue culture system, exploring a combination of plant growth regulators (PGRs) for Bougainvillea regeneration from in vitro stem segments. The Murashige and Skoog (MS) medium supplemented with indole-3-butyric acid (IBA), 6-benzylaminopurine (6-BA), and 1-naphthlcetic acid (NAA) was employed. The optimal sterilization of Bougainvillea stem segments involved a 30 s treatment with 75% alcohol and 10 min with 1% NaClO. The synergistic effect of 0.1 mg·L−1 of NAA and 2.5 mg·L−1 of 6-BA maximized the shoot sprouting frequency, while 2.5 mg·L−1 of 6-BA and 0.1 mg·L−1 of NAA produced the maximum shoots. Furthermore, 1.5 mg·L−1 of IBA and 0.1 mg·L−1 of NAA induced the highest rooting levels. This work demonstrates the successful adaptation of a greenhouse environment to efficiently regenerate plants in vitro from stem segments. This approach allows for the mass production of Bougainvillea glabra ‘New River’.

Production of quality planting material in commercial banana cvs. Rasthali (Silk, AAB) and Neypoovan (Neypoovan, AB) through farmer-friendly macropropagation technique and their field evaluation

Macropropagation is a low cost farmer friendly alternative to tissue culture propagation to cater to the farmers’ need for quality planting material at an affordable cost. Attempts were made to study the effect of age and hot water treatment on macropropagation of cvs. Rasthali (AAB, Silk) and Neypoovan (AB, Neypoovan) in pot and bed methods. Age of the sucker, hot water treatment and propagation method played a vital role on shoot bud production in both the test cultivars. In cv. Rasthali, bed method produced maximum number of plants (50.5) in five months old suckers subjected to hot water treatment in 42.2 days but the same treatment produced only 24.7 plants under pot method. In cv. Neypoovan, five months old suckers subjected to hot water treatment resulted in 23.3 and 16.7 plants in bed and pot methods respectively. Therefore, five months old suckers with hot water treatment under bed method are more appropriate for macropropagation purpose in banana. Results of field evaluation indicated that macropropagated bananas were at par with control in respect of all plant parameters like plant height, bunch weight and fruit parameters like total soluble sugar (TSS) and acidity.

Growth response of sweet kaffir lime (Citrus hystrix) seeds due to kinetin and coconut water application using tissue culture technique

Sweet kaffir lime is one of Aceh’s local fruit crops, which is rarely found anymore. The problem of sweet kaffir lime cultivation is the difficulty of generative and vegetative plant propagation. The general objective of this research is to preserve the Acehnese sweet kaffir lime from extinction. One way that can be done is through plant propagation in tissue culture. The experiment was conducted in the Plant Tissue Culture laboratory of the Faculty of Agriculture, Universitas Malikussaleh. This research used a two-factor, completely randomized design, and ten replications. Two factors were tried: the concentration of kinetin (0, 1, and 2 mg/L) and the concentration of young coconut water (0, 15, 30 %). The results showed that increasing kinetin concentration could reduce shoot length, number of leaves, root length and slow the root emergence time of sweet kaffir lime in vitro. The best treatment was the 0 mg/L kinetin (without kinetin). Increasing coconut water concentration could increase shoot length, number of leaves, and root length of sweet kaffir lime in vitro. The best treatment was 30% coconut water concentration.

Changes in root metabolites and soil microbial community structures in rhizospheres of sugarcanes under different propagation methods.

Root metabolites and soil microbial community structure in the rhizosphere play critical roles in crop growth. Here, we assessed the efficiency of conventional and tissue culture propagation methods in modulating the soil health and microbiota in the rhizosphere of sugarcane (Saccharum officinarum L.) plants. The seeding canes were obtained using newly planted and two-year ratooned canes propagated by conventional (CSN and CSR) or tissue culture (TCN and TCR) methods. Changes in soil fertility, root metabolites and soil microbial community structure in the rhizosphere of sugarcane plants obtained using these canes were assessed. The activities of soil β-glucosidase and aminopeptidase, soil microbial biomass nitrogen, and abundances of soil beneficial microbes, both at phyla and genera levels, were significantly higher in the rhizosphere of sugarcane plants in TCN and TCR treatments than those in that of plants in CSN and CSR treatments. Furthermore, flavonoid and flavonol biosynthesis and alanine, aspartate and glutamate metabolism were significantly upregulated in the roots of TCR and TCN plants compared with those in the roots of CSN and CSR plants. These results suggest that the tissue culture propagation method is a sustainable method for sugarcane cultivation to improve soil fertility and health in sugarcane rhizosphere.

In vitro Propagation and Genetic Assessment by using Markers in Potato (Solanum tuberosum L.) cv Kufri Ganga

The purpose of this study was to determine the best types and concentrations of plant growth regulators to use during different stages of micropropagation, as well as to assess the genetic fidelity of tissue culture raised plants of a cv Kufri Ganga using reliable markers. According to the data on shoot bud initiation, a combination of 0.25 mgl-1 BAP and 0.5 mgl-1 Kinetin results in the greatest number of shoots per explant (3.4±0.11) in the shortest amount of time (3.2±0.09) days. The maximum number of in vitro shoots per shootlet (4.7±0.15) was recorded when auxins were combined with cytokinin (MS medium + 0.25 mgl-1 BAP + 0.01 mgl-1 IAA) for shoot proliferation. Root initiation was observed in (2.2±0.10) days on MS medium fortified with 1.5 mgl-1 NAA. The maximum number of in vitro roots per shoot (14.2±0.31) was observed when MS media was fortified with 2.0 mgl-1 IBA. Molecular markers were used to assess genetic fidelity in plants grown in vitro. Monomorphic DNA banding patterns were found in all tissue culture raised plants and mother plants, indicating true to type planting material. This protocol for tissue culture propagation, as well as testing its genetic fidelity, may be beneficial for the successful application of better conservation of germplasm.

Optimization of an Efficient Micropropagation Protocol and the Assessment of Genetic Fidelity of Solanum tuberosum L. cv Kufri Neelkanth

The aim of our experiment was to determine the optimal types and concentrations of plant growth regulators used during different stages of micropropagation and assess the genetic fidelity of tissue culture raised plants of a purple colored cv Kufri Neelkanth by using reliable markers. According to the shoot bud initiation data, it was found that utilizing a combination of 0.25 mg l-1 BAP and 0.5 mg l-1 Kinetin resulted in the greatest number of shoots per explant (3.5±0.12) within a timeframe of (3.3±0.08) days. The maximum number of in vitro shoots per shootlet (10.4±0.59) were recorded when auxins were used in combination with NAA (0.01 mg l-1 NAA) and Kinetin (0.25 mg l-1) for shoot proliferation. In vitro root initiation was observed in (2.1±0.07) days on MS medium fortified with 2.5 mg l-1 NAA. The maximum number of in vitro roots per shoots (13.0±0.55) were observed when MS media fortified with 2.5 mg l-1 IBA. Maximum 100% rooting was observed in all MS media supplemented with different concentrations of auxins. In vitro raised plants were assessed for genetic fidelity by using twenty RAPD primers (genetic markers). Out of twenty primers used only four primers produced amplification. DNA banding patterns of all tissue culture raised plants and mother plants were monomorphic showing true to type planting material. This protocol for tissue culture propagation along with testing its genetic fidelity could be useful for better conservation of germplasm and genetic transformation studies in potato.

Can endophytic microbial compositions in cane roots be shaped by different propagation methods.

In practical production, cane stems with buds are generally used as seed for propagation. However, long-terms cane stems only easily lead to some problems such as disease sensitivity, quality loss, etc. Recently, cane seedings, which are produced by tissue culture were used in sugarcane production, but few studies on cane health related to tissue culture seedings. Therefore, to evaluate the immunity and health of sugarcanes growing from different reproduction modes, the endophytic microbial compositions in cane roots between stem and tissue culture seedlings were analyzed using high-throughput techniques. The results showed that the endophytic microbial compositions in cane roots were significant differences between stem and tissue culture seedlings. At the genus level, Pantoea, Bacillus, Streptomyces, Lechevalieria, Pseudomonas, Nocardioides, unclassified_f__Comamonadaceae enriched as the dominant endophytic bacterial genera, and Rhizoctonia, Sarocladium, Scytalidium, Wongia, Fusarium, unclassified_f__Phaeosphaer, unclassified_c__Sordariom, unclassified_f__Stachybot, Poaceascoma, Microdochium, Arnium, Echria, Mycena and Exophiala enriched as the dominant endophytic fungal genera in cane roots growing from the tissue culture seedlings. In contrast, Mycobacterium, Massilia, Ralstonia, unclassified_f__Pseudonocardiacea, norank_f__Micropepsaceae, Leptothrix and Bryobacter were the dominant endophytic bacterial genera, and unclassified_k__Fungi, unclassified_f__Marasmiaceae, Talaromyces, unclassified_c__Sordariomycetes and Trichocladium were the dominant endophytic fungal genera in cane roots growing from stem seedlings. Additionally, the numbers of bacterial and fungal operational taxonomic units (OTUs) in cane roots growing from tissue culture seedlings were significantly higher than those of stem seedlings. It indicates that not only the endophytic microbial compositions in cane roots can be shaped by different propagation methods, but also the stress resistance of sugarcanes can be improved by the tissue culture propagation method.

Polyploid Induction and Identification of Rosa roxburghii f. eseiosa.

Rosa roxburghii f. eseiosa Ku is a variety of Rosa roxburghii, with two known genotypes: Wuci 1 and Wuci 2. The lack of prickle on the peel of R. roxburghii f. eseiosa makes it easy to pick and process, but its fruit size is small. Therefore, we aim to induce polyploidy in order to obtain a larger fruit variety of R. roxburghii f. eseiosa. In this study, current-year stems of Wuci 1 and Wuci 2 were used as materials for polyploid induction, which was carried out through colchicine treatment coupled with tissue culture and rapid propagation technology. Impregnation and smearing methods were effectively used to produce polyploids. Using flow cytometry and a chromosome counting method, it was found that one autotetraploid of Wuci 1 (2n = 4x = 28) was obtained by the impregnation method before primary culture, with a variation rate of 1.11%. Meanwhile, seven Wuci 2 bud mutation tetraploids (2n = 4x = 28) were produced by smearing methods during the training seedling stage. When tissue-culture seedlings were treated with 20 mg/L colchicine for 15 days, the highest polyploidy rate was up to 60%. Morphological differences between different ploidys were observed. The side leaflet shape index, guard cell length, and stomatal length of the Wuci 1 tetraploid were significantly different from those of the Wuci 1 diploid. The terminal leaflet width, terminal leaflet shape index, side leaflet length, side leaflet width, guard cell length, guard cell width, stomatal length, and stomatal width of the Wuci 2 tetraploid were significantly different from those of the Wuci 2 diploid. Additionally, the leaf color of the Wuci 1 and Wuci 2 tetraploids changed from light to dark, with an initial decrease in chlorophyll content followed by an increase. In summary, this study established an effective method for inducing polyploids in R. roxburghii f. eseiosa, which could provide a foundation for the breeding and development of new genetic resources for R. roxburghii f. eseiosa and other R. roxburghii varieties in the future.

Tissue culture and rapid propagation technology for Gentiana rhodantha.

Gentiana rhodantha is a perennial herb of the genus Gentiana (Tourn.) L. This study was novel in establishing a regeneration system of G. rhodantha using young leaves as explants on the Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs). The roots, stems, and leaves of G. rhodantha were used as explants. The effects of the optimal explant disinfection method, type of explant used, concentrations of PGRs added to the culture media on tissue culture, and rapid propagation of G. rhodantha were studied. The results showed that the optimal disinfection method for stems and roots consisted of disinfection using 75% ethanol for 50 s, followed by 4% sodium hypochlorite (NaClO) for 10 min. The optimal disinfection technique for leaves consisted of disinfection using 75% ethanol for 50 s, followed by 4% NaClO for 8 min. Root explant was the most suitable for inducing the callus of G. rhodantha on the MS medium supplemented with different PGRs. The optimal conditions for callus induction included 1.0 mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L α-naphthalene acetic acid (NAA). The callus induction rate using the root explant reached 94.28%. MS supplemented with 2.0 mg/L 6-BA and 0.1 mg/L NAA was the optimal medium for inducing adventitious shoots from the callus of G. rhodantha. The best medium for propagation and plantlets strengthening was MS supplemented with 0.8 mg/L 6-BA and 0.3 mg/L NAA, and the propagation index was 8.62. MS supplemented with 0.3 mg/L 3-indolebutyric acid was the best culture medium for inducing the rooting of adventitious buds, with the maximum rooting rate reaching up to 100%.

Morphological abnormalities in tissue culture-derived date palms (Phoenix dactylifera L.) in Qatar

Recently, the tissue culture (T.C.) propagation has played a major role in the massive expansion of date palm (Phoenix dactylifera L.) plantations by making available a large number of cloned plants. Nevertheless, even if the in vitro propagation is an effective and a rapid way of date palm production, it has been associated with the emergence of some growth problems. Morphological abnormalities have been observed in tissue culture- derived date palm. Dwarfism is the most common abnormality recorded in the Qatari dates plantations. Plants produced by tissue culture in “Nemishi” and “Lulu” are the most sensitive varieties to this problem. Moreover, the phenomenon of pollination failure locally known as “sheiss” was observed with the varieties Khalass and Kheneisi. These off type phenotypes appear in the plants maturity stage and are commonly detected in the field years after planting. Therefore, the detection of these abnormalities, at an early stage, should be performed. Since the genetic variation altered DNA methylation and gene expression are associated with these abnormal phenotypes, the molecular identification is inevitably the solution.

The Effect of Benzyl Amino Purine and 2,4-Dichlorophenoxyacetic Acid on in Vitro Propagation of Pranajiwa (Euchresta horsfieldii (Lesch) Benn.)

Pranajiwa (Euchresta horsfieldii (Lesch) Benn.) is an endangered medicinal plant known for its function as aphrodisiac, antidote, expectorant, and tonic. It also can be used to cure TBC. Research has found that pranajiwa is difficult to be propagated, but the tissue culture propagation method has never been applied to this species. This research is aimed to figure out the procedure to induce callogenesis of pranajiwa with the addition of Benzyl Amino Purine (BAP) and 2,4-Dichlorophenoxyacetic acid (2,4-D) and to determine the proper combination of BAP and 2,4-D to produce an optimal callus. MS-based medium with 25 combinations of BAP and 2,4-D has been used for the callus induction. Parameters observed in this research are callus appearing time, percentage of inducted calluses, and morphology observation, including the texture and color of the callus. Result shows that 90.67% calluses was successfully inducted. The optimal callus was obtained by combining BAP 1 ppm and 2,4-D 1,5 ppm with 5 days of appearing time, friable texture and green color.

High Efficiency Regeneration System from Blueberry Leaves and Stems.

The main propagation approach is tissue culture in blueberries, and tissue culture is an effective and low-cost method with higher economic efficiency in blueberries. However, there is a lack of stable and efficient production systems of industrialization of tissue culture in blueberries. In this study, the high-efficiency tissue culture and rapid propagation technology system were established based on blueberry leaves and stems. The optimal medium for callus induction was WPM (woody plant medium) containing 2.0 mg/L Forchlorfenuron (CPPU), 0.2 mg/L 2-isopentenyladenine (2-ip) with a 97% callus induction rate and a callus differentiation rate of 71% by using blueberry leaves as explants. The optimal secondary culture of the leaf callus medium was WPM containing 3.0 mg/L CPPU with an increment coefficient of 24%. The optimal bud growth medium was WPM containing 1.0 mg/L CPPU, 0.4 mg/L 2-ip, with which the growth of the bud was better, stronger and faster. The optimal rooting medium was 1/2 Murashige and Skoog (1/2MS) medium containing 2.0 mg/L naphthylacetic acid (NAA), with which the rooting rate was 90% with shorter rooting time and more adventitious root. In addition, we established a regeneration system based on blueberry stems. The optimal preculture medium in blueberry stem explants was MS medium containing 2-(N-morpholino) ethanesulfonic acid (MES) containing 0.2 mg/L indole-3-acetic acid (IAA), 0.1 mg/L CPPU, 100 mg/L NaCl, with which the germination rate of the bud was 93%. The optimal medium for fast plant growth was MS medium containing MES containing 0.4 mg/L zeatin (ZT), 1 mg/L putrescine, 1 mg/L spermidine, 1 mg/L spermidine, which had a good growth state and growth rate. The optimal cultivation for plantlet growth was MS medium containing MES containing 0.5 mg/L isopentene adenine, with which the plantlet was strong. The optimal rooting medium for the stem was 1/2MS medium containing 2.0 mg/L NAA, with which the rooting rate was 93% with a short time and more adventitious root. In conclusion, we found that stem explants had higher regeneration efficiency for a stable and efficient production system of industrialization of tissue culture. This study provides theoretical guidance and technical support in precision breeding and standardization and industrialization in the blueberry industry.

Establishment of Tissue Culture and Rapid Propagation System on <i>Mandevilla sanderi</i>

Establishment of Tissue Culture and Rapid Propagation System on <i>Mandevilla sanderi</i>

Design and Evaluation of a Smart Ex Vitro Acclimatization System for Tissue Culture Plantlets

One of the technological advancements in agricultural production is the tissue culture propagation technique, commonly used for mass multiplication and disease-free plants. The necessity for date palm tissue culture emerged from the inability of traditional propagation methods’ offshoots to meet the immediate demands for significant amounts of planting material for commercial cultivars. Tissue culture plantlets are produced in a protected aseptic in vitro environment where all growth variables are strictly controlled. The challenges occur when these plantlets are transferred to an ex vitro climate for acclimatization. Traditional glasshouses are frequently used; however, this has substantial mortality consequences. In the present study, a novel IoT-based automated ex vitro acclimatization system (E-VAS) was designed and evaluated for the acclimatization of date palm plantlets (cv. Khalas) to enhance their morpho-physiological attributes and reduce the mortality rate and the contamination risk through minimal human contact. The experimental findings showed that the morpho-physiological parameters of 6- and 12-month-old plants were higher when acclimatized in the prototype E-VAS compared to the traditional glasshouse acclimatization system (TGAS). The maximum plant mortality percentage occurred within the first month of the transfer from the in vitro to ex vitro environment in both systems, which gradually declined up to six months; after that, no significant plant mortality was observed. About 6% mortality was recorded in E-VAS, whereas 18% in TGAS within the first month of acclimatization. After six months of study, an overall 14% mortality was recorded in E-VAS compared to 41% in TGAS. The proposed automated system has a significant potential to address the growing demand for the rapid multiplication of tissue culture-produced planting materials since the plant survival rate and phenotype quality were much higher in E-VAS than in the conventional manual system that the present industry follows for commercial production.

Season, basal media and plant growth regulators effect in wood plant in vitro propagation: a comprehensive review

Wood plant in-vitro propagation is currently widely used and is not limited to the preparation of superior seeds but has been widely utilized for the production of phytopharmaceuticals and the production of a valuable and specific tree for the benefit of man. Studies focused on the influence of explant collection seasons, selection of basic media, and plant growth hormone. The results of the study showed the magnitude of the variables that must be considered in the propagation of tissue cultures even in the same species and types of cultures. The period of January and March is the best time for explant retrieval because it provides the best explant response in bud breakage, lowest browning, and best bud induction. The basic media of MS (Murashige & Skoog), as well as its various modifications and the basic medium of WPM (Wood Plant Medium), tend to be widely chosen as the basic media at various levels of subcultures in many woody plant species. The application of growth regulators shows a tendency to use BAP (benzyl amino purine) and NAA (naphthalene acetic acid) whose application is separately or in combination with a certain concentration according to the species and subculture phase. Other plant growth regulators (PGRs) that are also quite widely used are the IBA (indole butyric acid), IAA (indole acetic acid), and G3 (gibberellic acid) groups which are used in the multiplication and rooting stages. The information from the study can be used as basic information at the stage of optimizing tissue culture activities in different species, explanatory, and regeneration types. The success of the optimization stage will be used as an improvement to the previous woody plant tissue culture protocol so that optimization of success is achieved. In vitro propagation is always associated with the number of resources used, so efforts to improve in vitro protocols are a very important activity to evaluate that will lead to increased effectiveness to reduce resource inputs, and be able to realize low-cost wood plant tissue culture propagation.

Establishing an Efficient Regeneration System for Tissue Culture in Bougainvillea buttiana 'Miss Manila'.

Bougainvillea plants have important ornamental and cultural value, as well as applications, for example, in improving the ecological environment, sterilization and as anti-virals in medicines, etc. Among many varieties, Bougainvillea buttiana ‘Miss Manila’ is more popular in landscape applications in southern China because of its excellent qualities. However, because of the difficulty of sexual reproduction, low rooting rate of asexual propagation cuttings and low temperature tolerance, its large-scale development is limited. For easy, quick and mass multiplication of such cultivars, tissue culture technique can be put to use. In this study, nodal segments of ‘Miss Manila’ were used as explants, and a single-factor experiment was carried out with a variety of plant growth regulators and concentrations to establish and optimize a complete tissue culture propagation system. The results showed that the best disinfestation was 75% ethanol treatment for 0.5 min + 0.1% HgCl2 treatment for 9 min, and the survival rate was 86.83%. The best shoot initiation formula was MS + 6-BA (2.5 mg/L) + IBA (0.2 mg/L), and the induction rate was 95.14%. The best formula for shoot proliferation was MS + 6-BA (1.5 mg/L) + NAA (0.1 mg/L), and the proliferation coefficient was 3.13. The best rooting culture formula was 1/2 MS + IBA (2.0 mg/L), and the rooting rate was 81.97%. The survival rate for plantlet refining and transplanting was 93.33%. In this study, a relatively efficient regeneration system for the tissue culture and rapid propagation of Bougainvillea buttiana ‘Miss Manila’ was established to address the problems of rooting and transplanting of this variety, to benefit research on the industrialized production and regeneration systems of this variety, and to provide a reference for the tissue culture of other varieties of Bougainvillea or other woody plants.

Cultivar-specific markers, mutations, and chimerisim of Cavendish banana somaclonal variants resistant to Fusarium oxysporum f. sp. cubense tropical race 4

BackgroundThe selection of tissue culture–derived somaclonal variants of Giant Cavendish banana (Musa spp., Cavendish sub-group AAA) by the Taiwan Banana Research Institute (TBRI) has resulted in several cultivars resistant to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a destructive fungus threatening global banana production. However, the mutations in these somaclonal variants have not yet been determined. We performed an RNA-sequencing (RNA-seq) analysis of three TBRI Foc TR4–resistant cultivars: ‘Tai-Chiao No. 5’ (TC5), ‘Tai-Chiao No. 7’ (TC7), and ‘Formosana’ (FM), as well as their susceptible progenitor ‘Pei-Chiao’ (PC), to investigate the sequence variations among them and develop cultivar-specific markers.ResultsA group of single-nucleotide variants (SNVs) specific to one cultivar were identified from the analysis of RNA-seq data and validated using Sanger sequencing from genomic DNA. Several SNVs were further converted into cleaved amplified polymorphic sequence (CAPS) markers or derived CAPS markers that could identify the three Foc TR4–resistant cultivars among 6 local and 5 international Cavendish cultivars. Compared with PC, the three resistant cultivars showed a loss or alteration of heterozygosity in some chromosomal regions, which appears to be a consequence of single-copy chromosomal deletions. Notably, TC7 and FM shared a common deletion region on chromosome 5; however, different TC7 tissues displayed varying degrees of allele ratios in this region, suggesting the presence of chimerism in TC7.ConclusionsThis work demonstrates that reliable SNV markers of tissue culture–derived and propagated banana cultivars with a triploid genome can be developed through RNA-seq data analysis. Moreover, the analysis of sequence heterozygosity can uncover chromosomal deletions and chimerism in banana somaclonal variants. The markers obtained from this study will assist with the identification of TBRI Cavendish somaclonal variants for the quality control of tissue culture propagation, and the protection of breeders’ rights.