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Malus 'Baiyun' (registration no. 20210210), a new crabapple cultivar, was registered in 2021 by the Nanjing Forestry Unversity. However, the difficult rooting has greatly limited the production of high-quality M. 'Baiyun' in industrialization development. There is thus a pressing need to develop an organogenesis protocol for the in vitro propagation of M. 'Baiyun' to alleviate a shortage of high-quality M. 'Baiyun' seedlings. The results showed that choosing the apical bud in mid-March was an excellent explant material. To promote proliferation, the highest proliferation (6.27) of apical shoots was cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg·L-1 6-benzylaminopurine(6-BA) + 0.05 mg·L-1 indole-3-butyric acid (IBA). Subsequently, a 100% rooting rate, average number of roots per shoot of 6.2 and maximum length of roots of 4.96 cm were obtained on half-strength Murashige and Skoog (1/2 MS) medium with the application of 0.5 mg·L-1 naphthaleneacetic acid (NAA) or 0.6 mg·L-1 NAA + 0.7 mg·L-1 IBA. Additionally, thick and lateral roots were obtained with 0.6 mg·L-1 NAA + 0.7 mg·L-1 IBA. Our study is the first to establish an effective organogenesis protocol for new crabapple cultivars using stem segments.

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Ipomoea biflora L., commonly known as morning glory, is an herbaceous vine plant in the Convolvulaceae family and is widespread at low elevations in Taiwan and other East Asian countries. In September 2023, six I. biflora plants exhibiting small leaves, leaf yellowing, and shoot proliferation were observed in a vacant lot in Taiwan Agricultural Research Institute (TARI), Wufeng District, Taichung, Taiwan, representing 100% disease incidence in the area. All the symptomatic morning glory climbed onto Murraya paniculata L. (common jasmine orange) which however showed no similar symptoms. The total DNA (two samples for each plant) from leaf tissues of three symptomatic morning glory plants, two asymptomatic morning glory plants, and one asymptomatic common jasmine orange was isolated by the CTAB method (Fulton et al. 1995) and used for PCR with the universal primers, P1 (Deng and Hiruki 1991)/P7 (Schneider et al. 1995), to amplify a fragment containing partial 16S rDNA. Expected 1.8-kb bands were amplified from DNA extracted from all symptomatic plants, whereas no PCR product was detected from that of the asymptomatic I.biflora and M. paniculata plants. Six PCR products were cloned and sequenced in the Biotechnology Center DNA-sequencing facility at National Chung Hsing University, and one representative sequence was selected and deposited in GenBank. BLAST analysis revealed that the obtained 16S rDNA sequence (PP230905) shared 99.92% identity with the following phytoplasma strains: rapeseed phyllody phytoplasma (CP055264), plumbago auriculata leaf yellowing phytoplasma (MN239503), and aster yellows phytoplasma (MK992774), which all belong to the 16SrI subgroup. The query 16S rDNA sequence shares 99.84% identity with that of the 'Candidatus Phytoplasma asteris' reference strain (M30790), suggesting that the phytoplasma is a 'Ca. Phytoplasma asteris'-related strain. A virtual restriction fragment length polymorphism (RFLP) analysis was conducted using iPhyClassifier tool (Zhao et al. 2009), and the pattern derived from the 16S rDNA fragment of the I. biflora phytoplasma was identical (similarity coefficient 1.00) to the reference pattern of 16SrI, subgroup B (onion yellows phytoplasma OY-M; AP006628). Six total DNA samples from symptomatic plants were used as templates to amplify 842 bp secA sequences with SecAfor1 and SecArev3 primers (Hodgetts et al. 2008), and one representative sequence was deposited in GenBank. The partial secA sequence (PP263636) showed 98.22% identity with that of Trema levigatum witches'-broom phytoplasma (MW032212) that also belongs to the 16SrI group (Wan et al. 2021). Phylogenetic analysis of both 16S rDNA and secA confirmed I. biflora phytoplasma as 16SrI, subgroup B. Taken together, we concluded that the morning glory phytoplasma in this study was a 'Ca. Phytoplasma asteris'-related strain belonging to the 16SrI group. To the best of our knowledge, this is the first report of a phytoplasma-infected I. biflora in Taiwan, suggesting morning glory as a new natural host of 16SrI phytoplasmas, alongside other plants like roselle and citrus (Tseng et al. 2014; Feng et al. 2015).

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Sugarcane is used to produce sugar, ethanol, and other by-products, so it is considered one of the most important crops worldwide. Using temporary immersion systems for sugarcane micropropagation represents an alternative to reduce the labor force, increase plant development, and improve plant quality. Temporary immersion systems are semi-automated bioreactors designed for the large-scale propagation of tissues, embryos, and organs. These are temporarily exposed in a liquid culture medium under a specific time and immersion frequency. Using this protocol and a temporary immersion bioreactor, it is possible to achieve multiplication and rooting. The use of temporary immersion bioreactors improves the multiplication rate and the rooting of sugarcane, reducing the culture time, labor force, and reagents needed while maintaining high survival rates during acclimatization.

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In Mexico, wild agaves are important for the production of alcoholic beverages known as mezcal and pulque. However, the propagation of agave seeds and pups is not enough to satisfy the national demand. Temporary immersion systems represent an agave micropropagation alternative that reduces the labor force, increases development, and improves the quality of seedlings. The use of the SETIS™ bioreactor in A. marmorata and A. potatorum improves the multiplication rate and allows rooting. Additionally, this bioreactor reduces the culture time, labor force, and reagents needed while maintaining high survival rates during the acclimatization phase.

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Commercial plant tissue culture now primarily serves the ornamental horticulture industry. The main pillars of the commercial tissue culture business are scalability of production, cost reduction, limited labor involvement, high quality, and genetic homogeneity of propagated plants. Based on these requirements, the current protocol employs a partially immersed liquid culture medium supported by a flexible aluminum mesh raft with a wire stand to facilitate shoot organogenesis from the horizontally placed root explants and hold the plants upright for shoot multiplication and rooting of Limonium Misty Blue. It is a florist crop that is in high demand as both dried and fresh flower fillers in various floral decorations. The majority of cultivated Limonium or statice cultivars are heterozygous in nature and propagate commercially through in vitro propagation to cater to the huge demand for planting materials needed for flower production. This is the first protocol to describe direct shoot organogenesis from the roots in a liquid half-component of Murashige and Skoog's (1962) (MS) basal medium supplemented with 1.6 µM NAA and 1.1 µM BA. The regenerated shoots are multiplied and rooted at the same time on the raft in a MS-based liquid culture medium that included 0.44 µM BA and 1.07 µM NAA. In comparison to agar-gelled medium, plants cultured in liquid medium grow more quickly without any signs of hyperhydricity. In liquid medium, a clump of 4-5 shoots is formed from a single shoot explant within 4 weeks and are rooted simultaneously within 6 weeks. On average, seven explants may fit on each raft, so on average, 25 healthy plants are produced from a single bottle. The regenerated plants are easily hardened in the greenhouse, and using ISSR-based molecular markers, the genetic homogeneity of the randomly selected hardened plants can be determined.

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BACKGROUND: Citrus cultivar improvement via conventional breeding strategies is impeded by factors related to its reproductive biology. The orange is a hybrid between pomelo (Citrus maxima) and mandarin (Citrus reticulata). Among various orange cultivars, Valencia oranges have a bit of bitter tang mixed in with their sweetness, as Navel oranges are, the most widely cultivated citrus species, quite sweeter, and also don't contain any seeds. Tangelo mandarin orange cultivar is a hybrid of C. reticulata × C. maxima or × C. paradisi. OBJECTIVE: The present study was undertaken to optimize the hormonal composition of the media with regard to plant growth regulators for in vitro propagation of sweet orange cultivars from nodal segment explants. METHODS: The nodal segment explants were collected from three citrus cultivars, Washington Navel, Valencia and Tangelo. Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of growth regulators was used for shoot proliferation and root induction, and the optimum medium composition was assessed. The patent for Citrus Tissue Culture was obtained from the Office of Research Affairs, Haramaya University. RESULTS: The results indicate that the highest shoot response was recorded for Washington's navel with maximum shoot proliferation rate (99.75%), shoot number per explant (1.76), shoot length (10.70 cm), leaf number per explants (3.54) after three weeks of culture. In all experiments, no growth was observed for the basal MS medium. Phytohormone combinations of IAA (1.2 mg/L) and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Among the cultivars, there were significant differences for the highest rooting rate (81.255), root number (2.22), and root length (2.95 cm) variables for Washington Navel. The lowest rooting rate (48.45%), root number (1.47) and root length (2.26 cm) were observed for Valencia. The highest rooting rate (84.90%), root number per microshoot (2.22) and root length (3.05 cm) was on MS medium supplemented with 1.5 mg/L NAA. CONCLUSION: A comparison of different concentrations of IAA and NAA on root induction of microshoots from nodal segments of citrus cultivars demonstrated NAA was a more effective hormone than IAA.

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The known activities of cytokinins (CKs) are promoting shoot multiplication, root growth inhibition, and delaying senescence. 6-Benzylaminopurine (BAP) has been the most effective CK to induce shoot proliferation in cereal and grasses. Previously, we reported that in lemongrass (Cymbopogon citratus) micropropagation, BAP 10 µM induces high shoot proliferation, while the natural CK 6-(γ,γ-Dimethylallylamino)purine (2-iP) 10 µM shows less pronounced effects and developed rooting. To understand the molecular mechanisms involved, we perform a protein-protein interaction (PPI) network based on the genes of Brachypodium distachyon involved in shoot proliferation/repression, cell cycle, stem cell maintenance, auxin response factors, and CK signaling to analyze the molecular mechanisms in BAP versus 2-iP plants. A different pattern of gene expression was observed between BAP- versus 2-iP-treated plants. In shoots derived from BAP, we found upregulated genes that have already been demonstrated to be involved in de novo shoot proliferation development in several plant species; CK receptors (AHK3, ARR1), stem cell maintenance (STM, REV and CLV3), cell cycle regulation (CDKA-CYCD3 complex), as well as the auxin response factor (ARF5) and CK metabolism (CKX1). In contrast, in the 2-iP culture medium, there was an upregulation of genes involved in shoot repression (BRC1, MAX3), ARR4, a type A-response regulator (RR), and auxin metabolism (SHY2).

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Lingonberry (Vaccinium vitis-idaea L.) is an important and valuable horticultural crop due to its high antioxidant properties. Plant tissue culture is an advanced propagation system employed in horticultural crops. However, the progeny derived using this technique may not be true-to-type. In order to obtain the maximum return of any agricultural enterprise, uniformity of planting materials is necessary, which sometimes is not achieved due to genetic and epigenetic instabilities under in vitro culture. Therefore, we analyzed morphological traits and genetic and epigenetic variations under tissue-culture and greenhouse conditions in lingonberry using molecular markers. Leaf length and leaf width under greenhouse conditions and shoot number per explant, shoot height and shoot vigor under in vitro conditions were higher in hybrid H1 compared to the cultivar Erntedank. Clonal fidelity study using one expressed sequence tag (EST)-polymerase chain reaction (PCR), five EST-simple sequence repeat (SSR) and six genomic (G)-SSR markers revealed monomorphic bands in micropropagated shoots and plants in lingonberry hybrid H1 and cultivar Erntedank conforming genetic integrity. Epigenetic variation was studied by quantifying cytosine methylation using a methylation-sensitive amplification polymorphism (MSAP) technique. DNA methylation ranged from 32% in greenhouse-grown hybrid H1 to 44% in cultivar Erntedank under a tissue culture system. Although total methylation was higher in in vitro grown shoots, fully methylated bands were observed more in the greenhouse-grown plants. On the contrary, hemimethylated DNA bands were more prominent in tissue culture conditions as compared to the greenhouse-grown plants. The study conclude that lingonberry maintains its genetic integrity but undergoes variable epigenetic changes during in vitro and ex vitro conditions.

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Tamarillo (Solanum betaceum Cav.) is a subtropical solanaceous tree with increasing agronomic interest due to its nutritious edible fruits. Growing demand for tamarillo plants and fruits requires optimization of existing propagation methods and scaled-up systems for large-scale cloning of selected germplasm. Three in vitro protocols have been used to micropropagate tamarillo: (1) axillary shoot proliferation in a semisolid medium, (2) organogenesis, and (3) somatic embryogenesis procedures. Variables such as the age of the established shoot cultures and rooting treatments were also analyzed. The morphological and physiological quality of acclimatized plants derived from all the methodologies were compared, with seed-derived plants used as a control group. Overall, the results show that in vitro-derived plants have a similar development to seed-derived plants. Micropropagation by axillary shoot proliferation was highly efficient, with rooting rates above 80% in most treatments. Organogenesis induction was more effective from lamina explants using MS media with 2.0 mg·L-1 6-benzylaminopurine. Both organogenesis and somatic embryogenesis-derived plants were also morphologically and physiologically equivalent to seed and axillary shoot-derived plants. The specificities of each micropropagation method are discussed.

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Halophyte plants are potential resources to deal with the increasing soil salinity determined by climatic change. In this context, the present study aimed to investigate the germplasm conservation of Artemisia caerulescens collected in the San Rossore Estate (Pisa, Italy) through in vitro culture, biochemical properties, and the phytochemical composition of the volatile fraction of both in vitro shoots and different organs of wild plants (leaves, young and ripe inflorescences). The best medium tested for the shoot proliferation was MS, with the addition of 1 µM BA. Total polyphenol content and antioxidant activity were noticeable in both the inflorescences, while leaves and in vitro shoots showed lower amounts. Concerning the phytochemical investigation, the headspaces (HSs) and the essential oils (EOs) were characterized by oxygenated monoterpenes as the main chemical class of compounds in all samples, and with α- and ß-thujone as the major constituents. However, the EOs were characterized by noticeable percentages of phenylpropanoids (23.6-28.8%), with brevifolin as the unique compound, which was not detected in the spontaneous volatile emissions of the same parts of the wild plant. Good amounts of EOs were obtained from different organs of the wild plant, comprising between 0.17% and 0.41% of the young and ripe inflorescences, respectively.

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A decade ago, shoot proliferation symptoms (i.e., witches' broom) in carrots were believed to be the cause of 'Candidatus Phytoplasma' and Spiroplasma infection, yet in recent years this association appeared to have weakened, and a closer association was found with the yet-unculturable, psyllid-transmitted Gram-negative bacterium 'Candidatus Liberibacter solanacearum'. In Israel, carrots are grown throughout the year, yet shoot proliferation symptoms tend to appear only in mature plants and mostly in late spring to early summer. We hypothesized that factors such as plant age, temperature, and vector load, which vary during the year, have a critical effect on symptom development and examined these factors under controlled conditions. Here we show that young carrot seedlings are as prone as older plants to develop shoot proliferation symptoms after 'Ca. L. solanacearum' inoculation. Surprisingly, we found that the local 'Ca. L. solanacearum' haplotype was extremely sensitive to constant temperature of 30°C, which led to a significant reduction in bacterial growth and symptom development compared with 18°C, which was very conducive to symptom development. We have also found that inoculations with 10 or 20 psyllids per plant results in faster symptom development compared with inoculations with two psyllids per plant; however, the difference between vector loads in disease progress rate was not significant. These data provide important insights to the effects of plant age, growth temperature, and vector load on 'Ca. L. solanacearum' and its associated symptoms and further strengthen the notion that 'Ca. L. solanacearum' is the main responsible agent for carrot witches' broom in Israel.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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The aim of this work was to assess the regeneration capacity of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica cv. 'Jugana' from different types of explants under various hormonal treatments. The whole leaves, petioles, and internodal segments of in vitro plants were examined as explants. Several plant growth regulators (cytokinins and auxins) were evaluated for their ability to induce adventitious regeneration. Direct and indirect organogenesis was achieved under certain culture conditions in both species. The frequency of shoot regeneration was strongly dependent on concentrations of plant growth regulators in the induction media (L.kamtschatica 'Jugana') or concentrations of plant growth regulators in the induction media and type of explant (A. alnifolia var. cusickii). Results showed that leaves were not suitable explants for A. alnifolia var. cusickii. Both species were able to regenerate shoots from internodal segments and petioles. The highest induction of shoots was obtained on Murashige and Skoog (MS) medium enriched with 2 mg/L thidiazuron (TDZ) and 0.5 mg/L indole-3-butyric acid (IBA) for Amelanchier alnifolia and with 1 mg/L TDZ and 0.2 mg/L indole-3-acetic acid (IAA) for L. kamtschatica 'Jugana'. Obtained adventitious shoots were further proliferated in order to investigate their multiplication capacity. The multiplication of shoots was successful in all cultivars, with the best results reported in A. alnifolia var. cusickii (7.07 shoots/explant on average).

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Artificial intelligence (AI) models and optimization algorithms (OA) are broadly employed in different fields of technology and science and have recently been applied to improve different stages of plant tissue culture. The usefulness of the application of AI-OA has been demonstrated in the prediction and optimization of length and number of microshoots or roots, biomass in plant cell cultures or hairy root culture, and optimization of environmental conditions to achieve maximum productivity and efficiency, as well as classification of microshoots and somatic embryos. Despite its potential, the use of AI and OA in this field has been limited due to complex definition terms and computational algorithms. Therefore, a systematic review to unravel modeling and optimizing methods is important for plant researchers and has been acknowledged in this study. First, the main steps for AI-OA development (from data selection to evaluation of prediction and classification models), as well as several AI models such as artificial neural networks (ANNs), neurofuzzy logic, support vector machines (SVMs), decision trees, random forest (FR), and genetic algorithms (GA), have been represented. Then, the application of AI-OA models in different steps of plant tissue culture has been discussed and highlighted. This review also points out limitations in the application of AI-OA in different plant tissue culture processes and provides a new view for future study objectives. KEY POINTS: • Artificial intelligence models and optimization algorithms can be considered a novel and reliable computational method in plant tissue culture. • This review provides the main steps and concepts for model development. • The application of machine learning algorithms in different steps of plant tissue culture has been discussed and highlighted.

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Cytokinins (CKs) are a chemically diverse class of plant growth regulators, exhibiting wide-ranging actions on plant growth and development, hence their exploitation in agriculture for crop improvement and management. Their coordinated regulatory effects and cross-talk interactions with other phytohormones and signaling networks are highly sophisticated, eliciting and controlling varied biological processes at the cellular to organismal levels. In this review, we briefly introduce the mode of action and general molecular biological effects of naturally occurring CKs before highlighting the great variability in the response of fruit crops to CK-based innovations. We present a comprehensive compilation of research linked to the application of CKs in non-model crop species in different phases of fruit production and management. By doing so, it is clear that the effects of CKs on fruit set, development, maturation, and ripening are not necessarily generic, even for cultivars within the same species, illustrating the magnitude of yet unknown intricate biochemical and genetic mechanisms regulating these processes in different fruit crops. Current approaches using genomic-to-metabolomic analysis are providing new insights into the in planta mechanisms of CKs, pinpointing the underlying CK-derived actions that may serve as potential targets for improving crop-specific traits and the development of new solutions for the preharvest and postharvest management of fruit crops. Where information is available, CK molecular biology is discussed in the context of its present and future implications in the applications of CKs to fruits of horticultural significance.

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Magnolia lucida (Magnoliaceae) is classified as an endangered species by the International Union for Conservation of Nature. It has high commercial value owing to its attractive tree shape and flowers. We adopted an excellent genotype of M. lucida as the parent material and established a mini-cut orchard through grafting to provide trunk shoots explants over the long-term. Optimal sterilization was achieved using a combination of 75% ethanol for 30 s, one percent benzalkonium bromide for five minutes, and 0.1% mercuric chloride for five minutes. Modified Murashige and Skoog medium (ML) was the optimal medium for the growth of M. lucida. Addition of one mg/L of 6-benzyl adenine (BA) and 0.05 mg/L of α-naphthaleneacetic acid (NAA) to the medium increased the shoot induction rate to 95.56%, and the ML medium containing 0.4 mg/L BA and 0.04 mg/L NAA achieved the maximum multiplication rate (284.56%). Dark treatment for seven days, followed by continuous light treatment could better resolve the challenge of difficult rooting in M. lucida plants. Using random amplified polymorphic DNA and inter simple sequence repeat markers, we confirmed the genetic uniformity and stability of the regenerated plants. Our protocol should be helpful for the propagation and conservation of this endangered plant.

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A number of Aloe species are facing an extremely high risk of extinction due to habitat loss and over-exploitation for medicinal and ornamental trade. The last global assessment of Aloe peglerae Schönland (in 2003) ranked its global conservation status as 'endangered' with a decreasing population trend. In the National Red List of South African Plants, the extremely rapid decline of this species has resulted in its conservation status being elevated from 'endangered' to 'critically endangered' based on recent or new field information. This dramatic decline necessitates the development of a simple, rapid and efficient micropropagation protocol as a conservation measure. An in vitro propagation protocol was therefore established with the regeneration of 12 shoots per shoot-tip explant within 8 weeks using Murashige and Skoog (MS) medium supplemented with 2.5 µM meta-topolin riboside (an aromatic cytokinin). The rooting of the shoots with a 100% frequency on half-strength MS medium without any plant growth resulted in additional six shoots produced per cultured shoot. The resultant plantlets were successfully acclimatized with a 100% survival frequency after 6 weeks. Overall, the developed protocol can result in the production of 3906 transplantable shoots that are ready for rooting per annum from a single shoot-tip explant. It is simple and efficient for seedling production in the ex situ cultivation and conservation of the endangered A. peglerae.

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There are more than nine thousand cultivars of Hibiscus rosa-sinensis L., with a series of flowers with shapes, colors and new cultivars continues as generated through both traditional and modern breeding techniques. In this study, advanced biotech methods of in vitro culture have been used to identify a technique for the efficient mass multiplication of H. rosa-sinensis 'White Butterfly', using phenyl urea, N-(2-Chloro-4-pyridyl)-N'-phenylurea (4-CPPU). For the first time, the effects of 4-CPPU for stimulating axillary shoot proliferation and multiple shoot regenerations from nodal explants were evaluated, and the optimal nutrient media deduced. From the diverse concentrations as 0.1, 0.5, 2.5, 5.0 & 10.0 µM of 4-CPPU, the highest frequency of shoots was recorded at 2.5 µM supplied in Murashige and Skoog (MS, pH-5.8) medium. After eight-weeks of culture, on an average of 6.7 shoot were obtained on this media with shoot heights of 4.2 cm from each explant. With the involvement of 0.5 µM-IBA (indole-3-butyric acid) in MS medium the regenerated shoots were rooted and followed by successful acclimation to ex vitro conditions. The ploidy consistency among the micro-plants was analyzed using flow cytometry and compared with ex vitro grown plants. No differences in the ploidy levels were observed among the 4-CPPU induced plants, when compared with the donor plants.

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Symptoms of excessive shoot proliferation were observed in the Njallani cultivar of small cardamom accompanied by stunting of stalks with fewer degenerated capsules at Nedumkandam Panchayat of Idukki district of Kerala in 2017. Five symptomatic Elettaria cardamomum shoot proliferation (ECSP) plant samples were collected and processed for DNA extraction and PCR assays utilizing universal phytoplasma 16S ribosomal-specific primers pair, P1/P7 followed by R16F2n/R16R2. Sequence comparison analysis of the R16F2n/R16R2 region of 16SrRNA gene showed 100% sequence identity with the 'Candidatus Phytoplasma australasia'- related strain. Phylogeny and virtual RFLP analyses of 16S rRNA gene sequences confirmed the association of 'Ca. P. australasia' strain subgroup D with ECSP disease. The association of 16SrII group was further established and validated by amplifying phytoplasma-specific multilocus candidate genes by utilizing specific primers of secA, secY, SAP11, and tuf genes. The multilocus gene sequence comparison analysis again confirmed the association of 'Ca. P. australasia' with the ECSP phytoplasma isolate. This is the first report of phytoplasma association with small cardamom.

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The effect of plant growth regulators on shoot proliferation from shoot tip explants of Ajuga multiflora was studied. The highest number of shoots (17.1) was observed when shoot tip explants were cultured on Murashige and Skoog (MS) medium fortified with 8.0 µM 6-Benzyladenine (BA) and 2.7 µM α-naphthaleneacetic acid (NAA). The mean number of shoots per explant was increased 1.6-fold in liquid medium as compared with semi-solid medium. Maximum rooting (100 %) with an average of 7.2 roots per shoot was obtained on MS basal medium. Rooted plantlets were successfully acclimatised in the greenhouse with 100 % survival rate. Composition of carotenoids, fatty acids and tocopherols was also studied from leaves of greenhouse-grown plants and in vitro-regenerated shoots of A. multiflora. The greatest amounts of carotenoids, fatty acids and tocopherols were obtained from leaves of in vitro-regenerated shoots cultured on MS basal medium, followed by leaves of greenhouse-grown plants and leaves of in vitro-regenerated shoots cultured on MS basal medium with 2.0 µM BA or thidiazuron. The most abundant carotenoid in A. multiflora leaves was all-E-lutein (89.4-382.6 µg g-1 FW) followed by all-E-ß-carotene (32.0-156.7 µg g-1 FW), 9'-Z-neoxanthin (14.2-63.4 µg g-1 FW), all-E-violaxanthin (13.0-45.9 µg g-1 FW), all-E-zeaxanthin (1.3-2.5 µg g-1 FW) and all-E-ß-cryptoxanthin (0.3-0.9 µg g-1 FW). α-Tocopherol was the predominant tocopherol in A. multiflora leaves. Linolenic acid (49.03-52.59 %) was detected in higher amounts in A. multiflora leaf samples followed by linoleic acid (18.95-21.39 %) and palmitic acid (15.79-18.66 %).

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Somatic embryogenesis is a powerful tool both for cloning and studies of genetic transformation and embryo development. Most protocols for somatic embryogenesis induction start from zygotic embryos or embryonic-derived tissues which do not allow the propagation of elite trees. In the present study, a reliable protocol for somatic embryogenesis induction from adult trees of strawberry tree is described. Leaves from in vitro proliferating shoots were used to induce somatic embryo formation on a medium containing an auxin and a cytokinin. Somatic embryos germinated in a plant growth regulator-free medium.

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