Plant Cell, Tissue and Organ Culture最新文献

Potato is an important part of the traditional Norwegian diet, and the crop faces several challenges with respect to pests and diseases, as well as the increasingly challenging changes in climate. Genome editing may provide tools to improve the resilience of Norwegian potato cultivars to new climate challenges. We have altered the skin colour of two potato cultivars, ‘Desirée’ and ‘Nansen’ from red to yellow, as a proof-of-concept for the use of CRISPR/Cas9 in a Norwegian cultivar. Our method has involved the use of protoplasts and we have grown the regenerants for three successive clonal tuber generations to evaluate the stability of the edited plants over time and under varying temperature conditions in contained rooms in a greenhouse. We found that the protoplast method is well suited to achieving CRISPR/Cas9 applications. The results show that the yellow skin is consistent over the three generations of tuber propagation. We found some suspected somaclonal variation in the protoplast regenerants. Some of the variation which we observed under high temperatures (up to nearly 40ºC) during the second growth cycle, disappeared when cultivated under lower temperatures in the third cultivation cycle.

A microponic system (MP system), a propagation system combines micropropagation and hydroponic that, has been studied on some flower and fruit plants; however, the MP system has not yet been tested on herbal plants, especially Jasminanthes tuyetanhiae (Apocynaceae, Asclepiadoideae). This study used the MP system with nylon film (A4 cover nylon) to evaluate rooting, abnormal developmental phenomena, antioxidant enzyme activities, secondary metabolites of plantlets, and subsequent growth. Results showed that 2-cm shoots cultured on a micropropagation system obtained a rooting rate of about 50–60%, and plantlets recorded abnormal phenomena reaching 85% consisting of 22.33% vitrification, 19.33% leaf abscission, 43.33% callus at the root and only 15% plantlets without abnormalities. In contrast, the MP system pre-treated with 0.5 mg/L indole butyric acid (IBA) and supplemented with 4 mg/L silver nanoparticles (AgNPs) obtained 100% rooting and improved plantlet quality. Furthermore, the plantlets developed using the MP system recorded a 2-fold reduction in vitrification and leaf abscission compared to those using the micropropagation system. A reduction in callus formation and increased antioxidant activities were also noted. The acclimatization of plantlets derived from the MP system pre-treated with 0.5 g/L IBA and 4.0 g/L AgNPs was higher than the conventional treatments.

The cell wall rebuilding is one of the first stage of protoplast development that enables further mitotic divisions and differentiation. Therefore, this work focuses on the comparison of the cell wall regeneration in the parental protoplasts of Fagopyrum tataricum, F. esculentum and the F. tataricum (+) F. esculentum hybrids, which are promising materials in terms of future breeding and research programmes. It is worth emphasizing that the preparation of buckwheat hybrids using electrofusion was described for the first time. The results indicate that cell wall rebuilding exhibited a common mechanism for parent protoplasts and the heterokaryon as all analysed cell wall components recognising arabinogalactan proteins (JIM13, JIM16), extensin (JIM20), xyloglucan (LM25) and pectins (LM20, LM5, LM6) were detected during the process of wall regeneration. However, there were certainly differences in the spatio-temporal appearance or disappearance of individual epitopes during the 72 h of the cell culture, which have been discussed in the paper.

Sugars, hormones and plant cell wall components regulate many aspects of somatic embryogenesis which requires detailed investigations of the process. In the present study, we investigated the histology, chemical composition of the cell wall, sugar and nitrogen metabolism, and hormonal profile associated with the induction and formation of embryogenic calluses in Passiflora edulis Sims. Zygotic embryos were cultured in induction medium supplemented with 72.4 µM dichlorophenoxyacetic acid and 4.4 µM 6-benzyladenine for 30 days. The zygotic embryo consisted of a uniseriate protoderm and an undifferentiated mesophyll. After 20 days, globular-stage somatic embryos were observed at the periphery of embryogenic calluses. During callus formation, the cell wall of dividing cells showed high levels of methyl-esterified homogalacturonan (HGs) epitopes, recognized for JIM7 antibody. Epitopes of heteromannans (LM21) were identified only in the initial explants, whereas epitopes of xyloglucans (LM15) appeared throughout the morphogenetic process. Cells undergoing transdifferentiation in embryogenic calluses exhibited a drop in epitopes recognized for JIM7 and LM15 and absence of JIM5 labeling. Lipids and proteins were metabolized during early somatic embryogenesis. In contrast, hexose, starch, and amino acid concentrations increased. Hormonal dynamics were also altered: while indole-3-acetic acid and cytokinins decreased during the embryogenic process, jasmonic acid and ethylene showed the opposite trend, indicating a possible role of these stress hormones in the early somatic embryogenesis of passion fruit. These results shed light on the structural and physiological aspects of passion fruit somatic embryogenesis which may help optimize regeneration via somatic embryogenesis of this important commercial passion fruit species.

Ilex paraguariensis St. Hil. is cultivated in South America to prepare a tea-like infusion with pharmacological properties. Its recalcitrant character has hindered the development of a suitable method for propagating selected genotypes. This study aimed to produce in vitro plantlets from axillary shoots of adult plants using a temporary immersion bioreactor. During the elongation phase, the impact of ammonium and nitrate concentration on Murashige and Skoog quarter-strength (¼MS) formulation, macronutrient uptake, hormone supplementation, and explant density (5–30 explants) were analysed. In addition, during the induction and expression stages of root formation, the effect of indole-3-butyric acid (IBA), cadaverine, quercetin, and chlorogenic acid supplementation was studied. As a result, we propose a protocol for in vitro plantlet production of I. paraguariensis adult plants in bioreactors. Twenty-day-old stem segments established in semisolid ¼MS medium plus sucrose 30 g·L^− 1 and indole-3-acetic acid (IAA) 0.5 µM are transferred to the elongation phase during 40 days in 300 cc BIT bioreactor (20 explants per flask) containing 100 mL of ¼MS modified with double ammonium and nitrate content and N6-benzyladenine (BA) 20 µM. Then, shoots longer than one centimetre are removed from the explant and placed into a rooting medium consisting of ¼MS with sucrose 30 g·L^− 1 and supplemented with IBA 7.5 µM, cadaverine 20 µM, quercetin 20 µM, and chlorogenic acid 20 µM. Finally, the in vitro 30-day-old plantlets are acclimatised on 150 cc pots filled with non-sterile composted pine bark and controlled-release micro-fertiliser. An inter-simple sequence repeat assessment revealed no difference between in vitro plantlets and mother plants.

This study explored the impact of sodium chloride (NaCl) elicitation on the accumulation of primary and secondary metabolites and the oxidative stress responses of Inula crithmoides L. (golden samphire) in vitro shoot cultures. Elicitation involved applying different concentrations of NaCl (0, 50, 100, and 200 mM) for 4 weeks. This was followed by assessing its impact on plant growth, physiological parameters (pigments, hydrogen peroxide content, total soluble sugars and proteins, and proline), and secondary metabolism (phenylalanine ammonia-lyase activity, shikimic acid, phenolics, flavonoids, and hydroxycinnamic acids) in the shoots. The extracts were also analysed using high-performance liquid chromatography (HPLC). The NaCl elicitation did not affect shoot growth but increased physiological functions such as photosynthesis and oxidative stress management under moderate salinity levels. In addition, NaCl treatments increased the synthesis of soluble sugars and proteins, particularly proline, as well as bioactive phenolics such as gentisic acid, chlorogenic acid, 4-hydroxybenzoic acid, luteolin-7-O-glucoside, and naringenin-7-O-glucoside. The NaCl elicitation in golden samphire shoot cultures offers a significant method for enhancing the production of important nutritional and bioactive compounds. This underscores the species’ potential for cultivation in saline environments and provides valuable prospects for its utilization in the health and nutrition sectors.

Organic acids, the important flavor compounds in grape berries, are altered by temperature during berry development. To mitigate potential interference encountered in field experiments, we employed in vitro culture techniques to investigate the impact of ambient temperature (AT) on organic acid metabolism at various developmental stages. The results showed that for the young berry (YB), organic acid accumulation rose gradually below 35 °C, but at 40 °C, tartaric and malic acid were significantly inhibited. In comparison to temperatures below 35 °C, ripening berry (RB) exhibited accelerated degradation of malic acid and an increase in tartaric acid content at 40℃. Notably, the enzymes activity of NADP-malate dehydrogenase (NADP-MDH) and NADP-isocitrate dehydrogenase (NADP-IDH) exhibited a significant increase, whereas there was a substantial decrease in NADP malic enzyme (NADP-ME) activity. AT altered the expression of organic acid metabolism genes, exhibiting distinct patterns between YB and RB. At 40 °C, most genes were suppressed; however, the levels of tartaric acid synthesis genes increased during the RB stage, consistent with the rise in tartaric acid content. The correlation analysis revealed that the transport of organic acids plays a pivotal role in the alteration of grape’s organic acid content induced by AT. Our study uncovered ambient temperature’s impact on grape organic acid metabolism via in vitro experiments, providing a guidance for organic acid biosynthesis regulation in grape berry.

Abstract

Clonal breeding programs of Pinus maximinoi require the establishment of a robust somatic embryogenesis (SE) protocol to produce enough cell lines to accelerate the effective continuous deployment of elite planting stocks to research and commercial compartments. Somatic embryogenesis was induced from immature zygotic embryo explants enclosed in megagametophytes of P. maximinoi collected from two plantations located in different climatic conditions. Cones were collected during the winter months from July to August and the influence of seed family, cone collection date and culture medium formulation, with emphasis on the organic and inorganic nitrogen supply, were studied. Ammonium to nitrate molar ratios of 1:1 and 1:2 in modified Litvay’s medium (mLV) produced the highest numbers of extrusions, while a 1:4 ratio mostly produced unhealthy, non-embryogenic extrusions. The formation of a tissue showing a rapidly-proliferating, spiky morphotype was produced in a medium supplemented with 1.5 g/L of L-glutamine. Morphologically advanced cultures with nodular structures were produced in megagametophytes from both plantations in a 1:2 NH₄⁺:NO₃⁻ medium regardless of L-glutamine supplementation levels. The optimal medium for P. maximinoi SE induction contained a 1:2 NH₄⁺:NO₃⁻ molar ratio with 1.5 g/L L-glutamine. The synergy between the molar ratio of NH₄⁺:NO₃⁻ and L-glutamine resulted in the highest numbers of extrusions. The overall inductive competence window for somatic embryogenic response in P. maximinoi was determined to be from the second week of July to the first week of August for both plantations. The “peak” period was in the fourth week of July 2022. The success of the SE technology in P. maximinoi seed families is determined by the optimal inductive competence window of the immature megagametophytes enclosing zygotic embryos and the chemical composition of the induction medium in terms of the ammonium to nitrate molar ratio and the concentration of the L-glutamine used.

Abstract

Blepharispermum subsessile DC. is well known for its ethnomedicinal values. This plant has been used traditionally for the treatment of various diseases. Due to over-exploitation, habitat destruction, and climate change B. subsessile has been included in the list as threatened plants. Hence urgent attention is needed for the protection and conservation of this endangered plant. This plant is found in ‘Gurudangar’, Odisha, India and it is declared as Medicinal Plant Conservation Areas for in situ conservation of this plant species along with few other medicinal plants. In vitro propagation is an extremely effective alternative to overcome the limitations of conventional propagation methods and to escalate the production of B. subsessile. Keeping this in view, the present study was envisaged to develop an efficient protocol for in vitro plant regeneration of B. subsessile from cotyledonary node explant. Multiple shoots were induced from the cotyledonary node on Murashige and Skoog’s (1962) (MS) medium supplemented with different types and concentrations of plant growth regulators. A combined effect of 2.0 mg/L meta-Topolin (mT) and 1.0 mg/L indole-3-acetic acid (IAA) when augmented with MS medium was evaluated as optimum for multiple in vitro shoot regeneration from cotyledonary node. Further, in vitro nodal segments were inoculated on different plant growth regulator supplemented medium for upscaling and mT (2.0 mg/L) was found to be best for such in vitro shoot proliferation. The in vitro shoots were rooted on ½ MS medium supplemented with 0.5 mg/L indole-3-butyric acid (IBA). The in vitro regenerated plants were successfully acclimatized in the small pots containing sterile garden soil and sand (1:1) followed by the transfer to larger pots containing garden soil. The genetic fidelity of in vitro regenerated plants was assessed and ascertained by ISSR markers. The phytochemical analysis and antioxidant activity of the in vitro regenerated plants vis-à-vis mother plant were also evaluated to find out the biochemical fidelity.

Abstract

Because of their shallow root system, drought stress is a major problem in potato cultivation. Due to climate change more severe drought periods are expected to occur in the vegetative growth phase of potato growth. Therefore, there is a great need for drought tolerant potato genotypes. Potato responds to drought stress in the field in various ways, including osmoregulation. Osmotic stress can be induced in vitro by adding an osmotic agent and thus lowering the osmotic potential of the medium. In this study, a new, cost-effective in vitro test system is presented, in which the osmotic agent can be gradually added after root formation to prevent an osmotic shock. This is achieved by using sieves as plant holders and liquid medium, which, allows an improved simulation of gradually drying soil. Responses to osmotic stress in four potato genotypes were analysed and an increase in proline under osmotic stress was detected. Moreover, genes of interest that were postulated to be linked to drought stress were shown by quantitative qRT-PCR to be regulated under osmotic stress. Furthermore, we showed that the content of sorbitol, which was used as osmotic agent, was 700- fold higher for ‘Eurostarch’ after seven days under osmotic stress and 1093- fold higher after 14 days, respectively, compared to control plants without sorbitol addition. Therefore, further investigations must show, whether it was taken up through the roots, is metabolised, stored or de novo synthesised by the potato plants.

Keypoints

The established novel in vitro test system for potato allows gradually increasing stress exposition of rooted plants. Sorbitol seems not an ideal osmotic agent as it is likely taken up.