Germination Frequency Research Articles

In vitro propagation, synthetic seeds production and clonal fidelity assessment of regenerants of endangered herb Rheum emodi

Rheum emodi, pharmaceutically important medicinal plant is mainly found in northern Himalayas. This herb has therapeutic properties such as anticancerous, antimicrobial, behaves like astringent and improves gastro related problems. All these factors contribute in immense decline of this herb in its natural habitat as it is excessively used by local people for medicinal as well as common eating purpose. We have optimized the efficient in vitro plant regeneration system through the callus from leaf explant and synthetic seed production for this herb. The culture medium used was Murashige and Skoog (MS) basal medium with the different concentration of phytohormones. Highest percentage of callus formation (84.44±0.27%) was observed in MS medium perforated with 2,4-D in combination with BAP. The maximum number of shoots (3.67±0.27) per explant was attained on MS medium with BAP (35.5 μM) and Kn (11.61 μM). For synthetic seed production, encapsulation was done in 3% of sodium alginate+ 50mM CaCl2. The highest survival and germination frequency of somatic embryos without storage period were observed as 78.9±1.33 and 66.7±0.58 respectively. The survival and germination rate of encapsulated embryos at 4 ͦC were significantly reduced as compared to non-stored somatic embryos. For ascertaining the clonal fidelity, 20 ISSR markers and 15 RAPD markers were assayed and employed to validate the true-to-type regenerants of Rheum emodi. Out of 15 RAPD and 20 ISSR markers, 7 markers and 15 markers produced distinct, clear and scorable bands. All the markers produced the monomorphic bands and no variation was detected among the micropropagated plants. Thus, the analysis of ISSR and RAPD patterns revealed that the bands were shared by both the in vitro raised plants and parent clump confirming the genetic stability. These markers proved to be a model tool for routine analysis of clonal fidelity of micropropagated plants prior to commercialization. These protocols could be beneficial in the future for rapid and mass multiplication or propagation of Rheum emodi and dissemination of synthetic or artificial seeds.

Identification and expression of strigolactone biosynthesis and signaling genes and the in vitro effects of strigolactones in olive (Olea europaea L.).

Strigolactones (SLs), synthesized in plant roots, play a dual role in modulating plant growth and development, and in inducing the germination of parasitic plant seeds and arbuscular mycorrhizal fungi in the rhizosphere. As phytohormones, SLs are crucial in regulating branching and shaping plant architecture. Despite the significant impact of branching strategies on the yield performance of fruit crops, limited research has been conducted on SLs in these crops. In our study, we identified the transcript sequences of SL biosynthesis and signaling genes in olive (Olea europaea L.) using rapid amplification of cDNA ends. We predicted the corresponding protein sequences, analyzed their characteristics, and conducted molecular docking with bioinformatics tools. Furthermore, we quantified the expression levels of these genes in various tissues using quantitative real-time PCR. Our findings demonstrate the predominant expression of SL biosynthesis and signaling genes (OeD27, OeMAX3, OeMAX4, OeMAX1, OeD14, and OeMAX2) in roots and lateral buds, highlighting their importance in branching. Treatment with rac-GR24, an SL analog, enhanced the germination frequency of olive seeds in vitro compared with untreated embryos. Conversely, inhibition of SL biosynthesis with TIS108 increased lateral bud formation in a hard-to-root cultivar, underscoring the role of SLs as phytohormones in olives. These results suggest that modifying SL biosynthesis and signaling pathways could offer novel approaches for olive breeding, with potential applicability to other fruit crops.

Valorizing Tree-Nutshell Particles as Delivery Vehicles for a Natural Herbicide.

The United States is a principal producer of tree nuts (almonds, pistachios, and walnuts), resulting in the generation of excess of tree-nutshell by-products each year, with few market outlets. A nutshell is an essential, lignocellulosic layer that protects a kernel (seed) from the environment during cultivation. The objective of this study was to develop nutshell by-products as herbicide delivery systems, which would not only enable sustainable weed control in fields but also increases nutshell value and reduce the cost of waste disposal. We recently identified a natural salicylaldehyde (SA) that emits volatiles with both herbicidal and antifungal properties. In this study, walnut shell particles saturated with 0.8 to 1.6 M SA were developed as delivery vehicles for SA to soil, which allowed for the controlled release of an SA fumigant for weed control. The pre- and post-emergent herbicidal efficacy of SA was investigated using model monocot (Lolium arundinaceum (Schreb.) Darbysh; turfgrass) and dicot (Brassica rapa var. pekinensis; Chinese cabbage) plants. We compared (1) the effects of different types of solvents for dissolving SA (dimethyl sulfoxide (DMSO) and ethanol (60%, v/v)), and (2) the effect of covering soil with plastic layers (i.e., soil pasteurization) or not covering soil during SA fumigation using nutshells. Results: In the pre-emergent herbicidal testing with the soil covered, the dicot plants exhibited levels of higher susceptibility to SA in DMSO emitted from nutshells when compared to the monocot plants. The seed germination frequencies in the dicots were 15% and 1% with 0.8 and 1.6 M SA, respectively, while those in the monocots were 32% and 18%, respectively, under the same test conditions. In the post-emergent herbicidal testing with the soil covered, the growth of both the monocot and dicot plants was completely prevented after 5 to 7 days of SA fumigation, resulting in the deaths of entire plants. It was noteworthy that in the post-emergent herbicidal testing, SA dissolved in ethanol (60%, v/v) completely disrupted the growth of the monocot and dicot plants as early as 3 days after SA emission from the nutshells, even without the soil being covered. Tree-nutshell particles could serve as effective SA delivery vehicles with controlled release capabilities for SA. The SA exhibited pre- and post-emergent herbicidal activities against the monocot and dicot plants at most growth stages. SA (0.8 and 1.6 M) dissolved in ethanol (60%, v/v) might exert a synergism for higher herbicidal activity after emission from nutshells. Since tree nuts capture/store a substantial amount of carbon over their life-cycles, the new and sustainable utility of using nutshells not only reduces carbon emissions but also valorizes tree-nut by-products, thus benefitting the tree-nut industry.

Plant regeneration from embryogenic callus-derived from immature leaves of Momordica charantia L

Bitter melon (Momordica charantia L.), a widely cultivated food and medicinal plant native to the world's subtropics and tropics, is a Cucurbitaceae rich in carotenoids. However, the low seed germination frequency and progeny variability associated with the production of this plant have a substantial impact on its growth and yield. These constraints affect the availability and exploitation of this crop, especially the fruits, which are rich in secondary metabolites such as β-carotene and α-carotene. In vitro regeneration would help overcome the obstacle linked to the germination of this plant and increase its yield and utilization. A reproducible in vitro organogenesis protocol was established using bitter melon embryogenic callus derived from immature leaf explants of in vivo grown seedlings and in vitro plantlets. Regeneration via callus was conducted on MSB5 media augmented with different plant growth regulator concentrations. The maximum frequency of callus formation (95.09 %) was produced in MSB5 media incorporated with 1.2 mg L−1 NAA augmented with 0.5 mg L−1 TDZ. MSB5 medium with no growth regulators was observed to be the most suitable for the shoot and root formation from the callus, producing a significantly high shoot percentage of 90.91 % and 21.53 shoots per explants, and the highest rooting frequency and root number of 88.92 % and 6.23 roots per explant, respectively, from leaf-derived callus of in vitro plantlets. The elongated plantlets had grown to a significantly higher average height of 12.20 cm on media added with 0.75 mg L−1 GA3. This reproducible method for regenerating bitter melon plantlets could facilitate mass multiplication, conservation, and commercial field production.

Deep learning-based high-throughput detection of in vitro germination to assess pollen viability from microscopic images.

In vitro pollen germination is considered the most efficient method to assess pollen viability. The pollen germination frequency and pollen tube length, which are key indicators of pollen viability, should be accurately measured during in vitro culture. In this study, a Mask R-CNN model trained using microscopic images of tree peony (Paeonia suffruticosa) pollen has been proposed to rapidly detect the pollen germination rate and pollen tube length. To reduce the workload during image acquisition, images of synthesized crossed pollen tubes were added to the training dataset, significantly improving the model accuracy in recognizing crossed pollen tubes. At an Intersection over Union threshold of 50%, a mean average precision of 0.949 was achieved. The performance of the model was verified using 120 testing images. The R2 value of the linear regression model using detected pollen germination frequency against the ground truth was 0.909 and that using average pollen tube length was 0.958. Further, the model was successfully applied to two other plant species, indicating a good generalizability and potential to be applied widely.

Protocol for plant regeneration from encapsulated somatic embryos of Citrus sinensis L.

BackgroundCitrus is an important fruit crop worldwide; artificial seeds are through encapsulation techniques of somatic embryos having many applications such as large-scale propagation and germplasm conservation. The aim of this study is the investigation of the viability of encapsulated citrus somatic embryos after different storage periods and to convert them into plantlets.ResultsCotyledonary-stage somatic embryos (5–7-mm size) regenerated from stigma explants of Washington navel orange (Citrus sinensis L.) were encapsulated individually in 3% sodium alginate. After different preservation periods (1, 2 or 3 months) at 10 °C, the encapsulated somatic embryos were cultured on Murashige and Skooge (MS) medium solidified with 7 g/l agar and supplemented with 50 g/l sucrose for germination. Percentage of regrowth, germination percentage, number of plantlets, plantlets height and number of leaves/shoot were recorded after different recovery periods. The germination percentages of encapsulated embryos were 90, 62.5 and 40% with storage for one, two and three months, respectively. Encapsulated somatic embryos preserved for 1 month developed the highest number of plantlets, while those preserved for 2 months developed the highest length of plantlets and the highest number of leaves/shoot after a recovery period of 10 months. Molecular analysis was performed of plantlets recovered from somatic embryos after preservation by encapsulation, and the results showed that the percentages of polymorphism were 7.7% with the two primers in all treatments.ConclusionsIt could be concluded that callus developed from stigma explants was able to regenerate indirect somatic embryogenesis after 3 months. Cotyledonary-stage somatic embryos of citrus were successfully preserved by encapsulation using 3% sodium alginate. Frequencies of germination of encapsulated somatic embryos increased with increasing the recovery period on the germination medium. A maximum recovery frequency of 60.8% was obtained from encapsulated somatic embryos cultured on germination medium for up to 10 months. Also, recovery frequency of 62.5% was noticed from encapsulated somatic embryos preserved for 2 months at 10 °C. In addition, results indicated that recovered somatic embryos obtained from encapsulated somatic embryos were able to convert to normal plantlets.

In vitro Conservation and Exploiting Polyembryonate Potential of Synthetic Seeds of Malaxis acuminata D. Don

Synthetic seeds were prepared in Malaxis acuminata by encapsulating shoot tip derived protocorm-like bodies (PLBs; 0.2-0.5 cm) in a hydrated gel capsule composed of calcium alginate matrix (sodium alginate; 3.5% and calcium chloride; 75 mM) in liquid Murashige and Skoog, 1962 (MS) medium. Synthetic seeds varied in physical features with the concentration of gelling agent and calcium chloride. In in vitro germination, the optimum response was achieved in MS medium fortified with BAP (1mgl-1); where synthetic seeds germinated with 100% frequency. Physically firm, globular, non-leaky, and self-breaking beads germinated efficiently which could be stored for up to 90 days at 4℃. At room temperature, synthetic seeds germinated with 50% frequency after 15 days and the germination frequency steadily reduced to 0% after 45 days of storage. Under in vivo conditions, synthetic seeds showed 80% bead-to-plant conversion in MS medium supplemented with antibacterial, antifungal agents and BAP (1mg/I). Plant Tissue Cult. & Biotech. 33(1): 9-15, 2023 (June)

Standardization of an In Vitro Seed Germination Protocol Compared to Acid Scarification and Cold Stratification Methods for Different Raspberry Genotypes

In raspberry (Rubus idaeus L.) breeding programs, seed double dormancy causes delayed and low germination rates. In this study, an in vitro germination protocol was developed to increase raspberry seed germination rate and decrease germination time. This protocol was optimized on the cultivars “Polka” and “Tulamagic”, specifically, their open-pollinated seeds, by applying two different cutting types (transverse, or longitudinal), followed by two different treatments (direct in vitro culture at 24 °C, or 2 weeks at 4 °C followed by in vitro culture at 24 °C). The best results for both cultivars were obtained when seeds were cut transversally and directly cultured in vitro at 24 °C (germination rate 73–78%, starting germination time 4 days after treatment). The optimized in vitro protocol was applied to 14 different combination crosses, observing a germination frequency around 87%, and speed in line with “Polka” and “Tulamagic”. These results were compared to those from the 14 cross combinations having undergone acid scarification (95% H2SO4 for 30 min) and cold stratification (3 months), which, in comparison, only yielded a 25% average germination rate. The optimized in vitro protocol favors high germination efficiency for all raspberry genotypes tested, and presents advantages for the standardization of germination time and reduction in the parental genotypes’ impact on germination rate.

Encapsulation of in vitro nodes of Stereospermum suaveolens DC. for propagation

Stereospermum suaveolens is a threatened medicinal tree that possesses winged seeds with low percent seed germination. To propagate this valuable species, synthetic seeds were prepared by encapsulation technique. Single in vitro nodes were excised from the in vitro shoot cultures and were utilized for encapsulation. Different gel matrix combinations were tested, of which 3% sodium alginate and 75mM CaCl2 were optimum in forming uniform synseeds. Utilizing this gel combination, different matrices were prepared and the frequency of percent germination of synseeds was recorded. The in vitro nodes encapsulated in MS+Kn8μM matrix, when placed in MS basal regenerative medium, resulted in maximum percent germination (100%) after 4 weeks whereas maximum shoot number (1.5±0.7) emerged from the encapsulated buds in WPM + Kn8μM matrix when placed in WPM + IBA 2μM static regenerative medium. Thus, the encapsulation technique can be utilized as an alternative method for the propagation of S.suaveolens.

Leaf-based induction of protocorm-like bodies, their encapsulation, storage and post-storage germination with genetic fidelity in Mokara Sayan × Ascocenda Wangsa gold

• First report on direct induction of PLBs Mokara Sayan × Ascocenda Wangsa gold. • Earliest and maximum PLB induction occurred on MS medium fortified with 3 mg L –1 TDZ. • Synthetic seeds were developed encapsulating PLBs with 3 % Na-alginate and 75 mM CaCl 2 . • Better storage of synthetic seeds up to 180 days was attained at 25°C in comparison to 4°C. • RAPD marker-based analysis ensured genetic integrity of synthetic seed-derived plantlets. An innovative and accelerated in vitro mass propagation protocol via direct induction of protocorm-like bodies (PLBs) from leaf sections of orchid hybrid Mokara Sayan × Ascocenda Wangsa gold was developed for the first time. Initially, leaf sections were transferred into Murashige and Skoog (MS) medium supplemented with two cytokinin sources namely, thidiazuron (TDZ) and zeatin with variable concentrations. The earliest (∼8 days) induction of PLBs with maximum number (34 PLBs cm –2 leaf section) and induction frequency (82.8 %) were recorded in MS medium fortified with 3 mg L –1 TDZ. The same medium formulation resulted in the highest growth rate (∼93.7 mg day –1 ) with maximum diameter of PLBs (3.4 mm) when compared with other treatments. Observations on proliferating PLBs under variable pressure scanning electron microscopy illustrated their distinctive growth phases wherein proliferating PLBs exhibited globular shape, typical epidermal texture with a rough surface that steadily became more wrinkled, consisting of basal absorbing hair-bodies and the apical zone forming shoot primordia. Stereo microscopic observations revealed gradual maturation of PLBs, initiation of leaf and shoot primordia. PLBs were encapsulated with calcium-alginate for short-term storage and germplasm exchange. Lower concentrations of sodium alginate (1 and 2 %) produced synthetic seeds that were soft to grip and extremely irregular in shape, whereas higher concentration (4 %) resulted in stiffness of synthetic seeds and reduced germination frequency. The most suitable gel matrix to form uniform spherical beads was optimized at 3 % sodium alginate and 75 mM calcium chloride. Synthetic seeds were effectively stored at 25 °C for up to 180 days. The germination efficiency of synthetic seeds gradually declined after storage at both 4 °C and 25 °C, with synthetic seeds stored at 25 °C being more tolerant than those stored at 4 °C. Even after 180 days of storage, the synthetic seeds that were stored at 25 °C exhibited 71.2 % germination frequency and ∼14 days to germination. Synthetic seeds stored at 4 °C deteriorated rapidly and degenerated completely within 180 days. Random amplified polymorphic DNA marker-based analysis carried out with randomly selected synthetic seed-derived plantlets showed 100 % monomorphism with a total 512 bands of molecular size ranging between 130—2100 bp, ensuring post-storage genetic fidelity.

Genome size and gas chromatography-mass spectrometry (GC-MS) analysis of field-grown and in vitro regenerated Pluchea lanceolata plants.

Pluchea lanceolata is a threatened pharmacologically important plant from the family Asteraceae. It is a source of immunologically active compounds; large-scale propagation may offer compounds with medicinal benefits. Traditional propagation method is ineffective as the seeds are not viable; and root sprout propagation is a slow process and produces less numbers of plants. Plant tissue culture technique is an alternative, efficient method for increasing mass propagation and it also facilitate genetic improvement. The present study investigated a three-way regeneration system in P. lanceolata using indirect shoot regeneration (ISR), direct shoot regeneration (DSR), and somatic embryo mediated regeneration (SER). Aseptic leaf and nodal explants were inoculated on Murashige and Skoog (MS) medium amended with plant growth regulators (PGRs), 2,4-dichlorophenoxy acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP) either singly or in combinations. Compact, yellowish green callus was obtained from leaf explants in 1.0 mg/l BAP (89.10%) added medium; ISR percentage was high, i.e., 69.33% in 2.0 mg/l BAP + 0.5 mg/l NAA enriched MS with 4.02 mean number of shoots per callus mass. Highest DSR frequency (67.15%) with an average of 5.62 shoot numbers per explant was noted in 0.5 mg/l BAP added MS medium. Somatic embryos were produced in 1.0 mg/l NAA fortified medium with 4.1 mean numbers of somatic embryos per culture. On BAP (1.0 mg/l) + 0.5 mg/l gibberellic acid (GA3) amended medium, improved somatic embryo germination frequency (68.14%) was noted showing 12.18 mean numbers of shoots per culture. Histological and scanning electron microscopic (SEM) observation revealed different stages of embryos, confirming somatic embryogenesis in P. lanceolata. Best rooting frequency (83.95%) of in vitro raised shootlets was obtained in 1.0 mg/l IBA supplemented half MS medium with a maximum of 7.83 roots per shoot. The regenerated plantlets were transferred to the field with 87% survival rate. The 2C genome size of ISR, DSR, and SER plants was measured and noted to be 2.24, 2.25, and 2.22 pg respectively, which are similar to field-grown mother plant (2C = 2.26 pg). Oxidative and physiological events suggested upregulation of enzymatic activities in tissue culture regenerated plants compared to mother plants, so were photosynthetic pigments. Implementation of gas chromatography-mass spectrometry (GC–MS) technique on in vivo and in vitro raised plants revealed the presence of diverse phyto-chemicals. The yields of alpha amyrin and lupeol (medicinally important triterpenoids) were quantified using high-performance thin-layer chromatography (HPTLC) method and enhanced level of alpha amyrin (2.129 µg g−1 dry wt) and lupeol (1.232 µg g−1 dry wt) was noted in in vitro grown leaf tissues, suggesting in vitro conditions act as a potential trigger for augmenting secondary metabolite synthesis. The present protocol represents a reliable mass propagation technique in producing true-to-type plants of P. lanceolata, conserving 2C DNA and ploidy successfully without affecting genetic homogeneity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13353-022-00727-7.

Potencial germinativo de sementes de pata-de-vaca de diferentes estádios de maturação sob temperaturas variadas

Abstract Bauhinia monandra Kurz has ornamental characteristics as the shape of its crown and the exuberance of its flowering, being and is widely cultivated in urban afforestation in Brazilian regions. The production of forest seedlings is essential not only to meet the demand of urban afforestation, but also to conserve forests. However, studies on B. monandra regarding the degree of fruit maturity on seed performance are scarce. Thus, the objective of the current work was to verify the germination potential of B. monandra seeds at different stages of maturation at five constant temperatures. The germination percentage, germination speed index (GSI), mean germination time (t), relative germination frequency, and mean germination speed (MGS) were evaluated. Initially, the mature seeds had a water content of 8.5% and the immature seeds of 68.3%. Mature seeds reached high germination percentages, above 96% for temperatures of 25, 30, and 35 ºC. The immature seeds demonstrated low germination percentages, reaching the highest percentage of 33% for the temperature of 25 ºC, followed by the temperatures of 30 ºC and 35 ºC, with respective percentages of 32% and 20%. Mature seeds demonstrated the highest GSI value (11.79) for the estimated temperature of 26.94 ºC and immature seeds the highest value of t (7.85) for the temperature of 26.56 ºC. It is concluded that mature seeds aggregate higher germination at an estimated temperature of 24.72 ºC and that immature seeds present unsatisfactory germination performance.

ENCAPSULATION OF BANANA SHOOT TIPS FOR In vitro SHORT-TERM STORAGE AND GERMPLASM CONSERVATION

The present investigation is focused on the effect of different concentrations of sodium alginate and calcium chloride treatment on banana shoot tip (G-9) encapsulation for the formation of artificial seeds. For this, different concentrations of sodium alginate and calcium chloride were checked for optimum encapsulation. In vitro culture shoot tips of bananas were encapsulated by using different concentrations of sodium alginate i.e. (1%, 2%, 3% and 4%) and calcium chloride (50 mM, 75mM, 100 mM and 150 mM). Further, the storage viability of encapsulated shoot tips was also examined. Encapsulated shoot tips were stored at two different temperatures i.e., 4℃ and 25℃. Results indicated that the best alginate concentration for encapsulation was found at 3% (w/v) and 100 mM calcium chloride. The beads were formed globular, firm and found suitable for handling and exhibited a germination frequency of 93.33% on Murashige and Skoog medium supplemented with BAP and IAA (5.0 + 1.0 mg/l, respectively). The encapsulated shoot tips showed different responses after storage at different temperatures. The multiplication frequency declined for both tested storage temperatures i.e., 4℃ and 25℃. Encapsulated shoot tips stored at 4℃ showed survival for longer storage with a regeneration frequency of 91.33%, whereas, capsules stored at 25℃ resulted in rapid deterioration within 90 days and exhibited a maximum multiplication frequency as 61.66%. This indicates that storage at low temperatures is more effective. This study could be applied as an alternative method of banana micropropagation as well as germplasm conservation.

Genome-wide characterization of chalcone synthase genes in sweet cherry and functional characterization of CpCHS1 under drought stress.

Cherries are one of the important fruit trees. The growth of cherry is greatly affected by abiotic stresses such as drought, which hinders its development. Chalcone synthase (CHS, EC 2.3.1.74) is a crucial rate-limiting enzyme in the flavonoid biosynthetic pathway that plays an important role in regulating plant growth, development, and abiotic stress tolerance. In the current study, three genes encoding chalcone synthase were identified in the genome of sweet cherry (Prunus avium L.). The three genes contained fewer introns and showed high homology with CHS genes of other Rosaceae members. All members are predicted to localize in the cytoplasm. The conserved catalytic sites may be located at the Cys163, Phe214, His302, and Asn335 residues. These genes were differentially expressed during flower bud dormancy and fruit development. The total flavonoid content of Chinese cherry (Cerasus pseudocerasus Lindl.) was highest in the leaves and slightly higher in the pulp than in the peel. No significant difference in total flavonoid content was detected between aborted kernels and normally developing kernels. Overexpression of Chinese cherry CpCHS1 in tobacco improved the germination frequency of tobacco seeds under drought stress, and the fresh weight of transgenic seedlings under drought stress was higher than that of the wild type, and the contents of SOD, POD, CAT, and Pro in OE lines were significantly increased and higher than WT under drought stress. These results indicate cherry CHS genes are conserved and functionally diverse and will assist in elucidating the functions of flavonoid synthesis pathways in cherry and other Rosaceae species under drought stress.

Coyote and porcupine spread Russian olive seeds through endozoochory

AbstractRussian olive (Elaeagnus angustifolia) is an invasive tree that has spread throughout much of the western United States. The mode of seed dispersal occurs by hydrochory and possibly by birds. Seed dispersal by frugivorous mammals has not been investigated. Between 15 October and 4 November 2020, we walked through Russian olive windbreaks in western North Dakota, USA, and surveyed for mammal scat, and found 10 coyote (Canis latrans) and 54 porcupine (Erethizon dorsatum) scats that contained intact Russian olive seeds. We subsequently evaluated the viability, germination frequency, and time to germination of seeds ingested by coyote and porcupine relative to un‐ingested control seeds harvested from trees at sites where we collected scat. Overall, Russian olive seeds that passed through mammal intestinal tracts had similar viability and equivalent (porcupine) or higher (coyote) germination frequency compared to controls. Additionally, coyote‐ingested seeds germinated earlier (time to germination was low) than controls, but porcupine‐ingested seeds were similar to controls. Thus, our data supports the idea that mammals may be agents of regional Russian olive seed dispersal.

Effects of Lead and Arsenic in Soils from Former Orchards on Growth of Three Plant Species.

Historical use of lead arsenate as a pesticide in former orchards of eastern Washington State (USA) has resulted in legacy lead (Pb) and arsenic (As) soil contamination. However, the impacts on plant growth in soils with residual Pb and As contamination have not yet been quantified. To this end, a comparative study of plant growth impacts was performed for native bluegrass (Poa secunda), invasive cheatgrass (Bromus tectorum), and buttercrunch lettuce (Lactuca sativa). Using standard plant growth protocols, germination frequency and biomass growth were measured over a wide range of Pb and arsenate concentrations, with maximum concentrations of 3400 and 790 mg kg−1 for Pb and As, respectively. Results indicated that only the biomass growth for all species decreased in soils with the highest concentrations of Pb and As in the soil, with no impacts on soils with lower residual Pb and arsenate concentrations. No impact on percentage of germination was observed at any soil concentration. These results can be used to determine site‐specific soil screening levels for use in ecological risk assessments for Pb and arsenate in soils. Environ Toxicol Chem 2022;41:1459–1465. © 2022 Battelle Memorial Institute. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

RNAi-mediated down-regulation of fasciclin-like proteins (FoFLPs) in Fusarium oxysporum f. sp. lycopersici results in reduced pathogenicity and virulence

Fusarium oxysporum f. sp. lycopersici (Fol) is majorly responsible for causing vascular wilt disease in tomato by blocking transpirational pull, thereby interfering and suppressing overall host immune response. This is mainly achieved by fungal invasion and colonization within the host, resulting in hyphal formation that instigates secondary infection response inside the plant. Earlier reports show the role of fasciclin-like proteins (FLPs) in cell-to-cell adhesions and signaling cascade. Moreover, deletion mutant of FLPs explained its role in development stages of Magnaporthe oryzae and Lentula edodes. Therefore, in present study, based on bioinformatic analysis, we have identified putative FoFLP genes encoding Fusarium-specific fasciclin like proteins. We have exploited the RNAi technology to analyse and understand role of these FoFLPs in virulence during wilt disease in tomato. Interestingly, the morphogenesis of fungal RNAi transformants of FoFLP1, FoFLP3, FoFLP4 and FoFLP5 showed significant reduction in spore count and spore germination frequency, thereby suggesting role of FoFLPs in conidiation. Furthermore, we have detected FoFLP1, FoFLP3, FoFLP4 and FoFLP5 specific siRNAs in the respective fungal transformants, using stem-loop RT-PCR and northern hybridization suggesting targeting of cognate FoFLPs transcripts. Moreover, the fruit invasion and plant infection assays using FoFLP fungal transformants showed late onset of disease with significant reduction in disease symptoms in the infected plants. Although the FoFLP-RNAi fungal transformants entered the host plant by penetrating root cortex, the infected plants showed minimum fungal colonization as a result of siRNA-mediated targeting of endogenously expressed FoFLPs. This further inhibited the propagation of fungal RNAi transformants within root cortex and affected its ability to cause secondary infection response, which led to reduced disease incidence and disease severity index. Altogether, our results show that FoFLPs might play an important role in conidiation and pathogenicity, and can serve as potent RNAi targets. Therefore, Host-Induced Gene Silencing (HIGS) can effectively be used to target FoFLPs in order to raise tomato transgenics resistant to Fusarium wilt.

M-brigde- and elicitor-assisted enhanced post-storage germination of Rauvolfia serpentina synthetic seeds, their genetic fidelity assessment and reserpine estimation

The present study reports an amelioration of synthetic seed germination efficiency with a simple intervention of filter paper (M-bridge) drenched in ½Murashige and Skoog liquid (½MSL) medium in Rauvolfia serpentina. Synthetic seeds were developed from in vitro shoot tips using 3% (w/v) sodium alginate and 75 mM calcium chloride. Media for synthetic seed gemination were tested in cotton bed drenched with sterile water (SW), cotton bed drenched with ½MSL medium, M-bridge drenched with ½MSL medium, and ½MS semisolid (½MSS) medium alone. Synthetic seeds inoculated on ½MSL or SW or ½MSS media, exhibited slower germination and inferior performance; however, with the involvement of M-bridge, fastest germination (5.6 ± 0.4 days) and maximum proliferation (4.8 ± 0.7 shoots with 42.8 ± 2.4 mm length, and 19.8 ± 2.7 leaves/synthetic seed after 42 days of inoculation) were recorded. Among the tested elicitors (silver nitrate, salicylic acid and sodium nitroprusside) at their different concentrations supplemented in ½MSL medium (with M-bridge), 50 µM silver nitrate resulted in 100% germination frequency and resulted in germination within 5 days. Further, the synthetic seeds were stored for 42 days at − 20 °C, 8 °C, and 25 °C. Maximum germination efficiency was obtained in case of seeds stored at 8 °C with respect to earliness in germination (6.9 ± 0.3 days), germination percentage (71.8 ± 3.4%) and subsequent proliferation (4.9 ± 0.6 shoots with 18.9 ± 0.9 mm length, and 8.9 ± 0.7 leaves/synthetic seed). Clonal fidelity analysis of the in vitro regenerants performed using RAPD markers exhibited monomorphic banding pattern. The reserpine content of the regenerants was found to be maximum following 42 days-storage at 8 °C (126.9 µg/g), followed by a substantial decline in the synthetic seed-derived regenerants following 42-days-storage at 25 °C (120.7 µg/g) or − 20 °C (121.8 µg/g).

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill.

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP + 1.4 µM GA3 enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11240-022-02254-z.

Synthetic Seed Production and Germplasm Conservation of Enicostema axillare (Lam.) raynal ssp. littoralis (Blume) raynal Using Nodal and Root Explants

In the present study, synthetic seed production of the medicinally important plant Enicostema axillare through nodal and root explants were attempted. Enicostema axillare plantlets were germinated through the production of synthetic seeds using in vitro nodal and root explants. Synthetic seed prepared from 3% Na+ and 100mm calcium chloride was the optimum concentration for uniform and compact synseed formation. Alginate beads containing in vitro derived nodal explants were successfully entrapped with 3% sodium alginate prepared in half-MS, quarter-MS, full-strength MS, and double-distilled water and 100 mM of CaCl2 solution. Synthetic seeds prepared from full strength MS medium exhibited the highest number of multiple shoots on benzyl amino purine (BAP) 2 mgl-1 and kinetin (KIN) 0.5 mgl-1 in combination with 2 mgl-1 GA3. The capsulated nodal explants germinated and produced multiple shoots of 15.0±1.87 at a high rate (87%) in 2 mgl-1 BAP and KIN 0.5 mgl-1 in combinations with 2 mgl-1 GA3 with a shoot length of 6.24 ±1.03 cm along with a root number of 28.4±5.50. Synthetic seeds stored at 18ºC were found to be favorable temperature for the preservation of synthetic seeds, and it increased seed germination frequency. The synseed nodal explants prepared using ¼ strength MS medium and stored at 18ºC were preserved for up to 80 days and more without losing germination ability (80%). The fully developed plantlets were hardened on vermiculite (100%) with a survival rate of 71%. This protocol could be used for the conservation of germplasm for long-term storage of E. axillare.