In Vitro Cellular & Developmental Biology - Plant最新文献

Wild bananas and their relatives are potentially utilized for pre-breeding due to their genetic diversity, disease resistance, and tolerance to abiotic stress, and other desirable traits. The embryonic suspension culture of wild bananas provides a means to harness this genetic diversity for banana genetic improvement. This paper elucidates the response of different subspecies M. acuminata (ssp. malaccensis, microcarpa, sumatrana, and breviformis) to the induction, growth, and behavior of suspension cultures and their regeneration into plantlets. Different subspecies exhibit varied responses starting from the embryogenic culture induction stage, culture proliferation, to plantlet formation. The highest competence for plant regeneration through somatic embryogenesis was found in ssp. malaccensis, followed by microcarpa, sumatrana, and breviformis. The wild banana embryogenic culture consists of somatic embryos, somatic embryo masses, proembryos, and proembryonic masses, and it proliferates through somatic embryo budding and proembryo proliferation. Maintenance and proliferation of suspension cultures were achieved through subculturing medium-sized cell aggregates (300 to 1000 µm). With an inoculum density of 0.3 g per 30 mL medium, the culture’s proliferation rate reached seven times within 25 d. Embryogenic cultures from the suspensions of ssp. malaccensis and microcarpa were capable of forming somatic embryos upon transfer to a semi-solid somatic embryo development medium and later developed shoots on a semi-solid plant regeneration medium, with conversion efficiencies of 35% and 17%, respectively.

The Turiaçu pineapple cultivar produces fruits of high organoleptic value but has few biotechnological studies on seedling production. However, conventional in vitro propagation can affect the photosynthetic potential of plants when transferred to the field, thus mitigating measures should be undertaken to solve this limitation, for example by decreasing carbohydrate concentration in the growth medium, adopting bioreactors of temporary immersion with forced ventilation, and using gas permeable membranes in the culture flask. The present work focused on evaluating the growth and development of plantlets from Ananas comosus [L]. Merr. cultivar Turiaçu, an important but neglected pineapple cultivar, under different sucrose concentrations and cultivation systems. For that, the impact of the photomixotrophic and photoautotrophic growth on morphophysiological responses of plants and survival during the ex vitro acclimatization was assessed. The plants were grown in four cultivation systems: permanent immersion system with sealed flasks (PIS-SF); permanent immersion system with natural ventilation (PIS-NV); single-flask temporary immersion bioreactors (TIS-PF); and twin-flasks temporary immersion bioreactors (TIS-RALM), combined with sucrose concentrations (0, 5.0, 15.0, and 30.0 g L⁻¹). The results indicate that Turiaçu plants have photoautotrophic potential in vitro, as the photochemical efficiency of the plants increased in cultivation systems with TIS –RALM gas exchange without the addition of sucrose. Furthermore, it also improved the performance and hardening of plants in ex vitro conditions, which constitutes a crucial step towards the diffusion of this cultivar.

Haploidization technique is the modern and advanced breeding technique used to enhance economically essential crops, including cucumber, to meet consumers’ preferences and needs worldwide. This study aimed to evaluate the effects of factors such as genotype, thermal pre-treatments, and nutrient medium combinations that are considered in haploid production via ovary culture in four commercial cucumber varieties. Four different induction media with four distinct maturation medium combinations and three F1 and one open-pollinated (Beith Alpha) cucumber varieties were used. All explants (cucumber ovaries’ slices) were inoculated in induction media and remained at 35 °C/3 d in the dark, then were transferred at 25 ± 1 °C 16-/8-h light/dark photoperiod for a further 9 d. The results of the study demonstrated that the embryo-like structure (ELS) formation rate was high in induction medium 2 which was successful in all varieties of Ptk40 (20%), Botanik (20%), Beith Alpha (16%), and Sardes (13.3%), respectively, compared to other induction medium combinations used in this study. Callus formation rates obtained from regeneration of medium 2 supplemented with 2.0 mg L⁻¹ 6-benzylaminopurine (BAP) and 0.5 mg L⁻¹ 1-naphthaleneacetic acid (NAA) were Ptk40 (65%), Sardes (60%), and Botanik (40%), and no callus was formed in Beith Alpha. Due to the findings of this study, the nutrient medium containing KIN and 2,4-D seems to be successful during induction of haploid embryo formation in cucumbers. One plantlet germinated but perished at early stage. The plantlet regeneration was observed to be low. Further studies are required to optimize the embryo regeneration medium composition due to the cucumber growing seasons, genotypes, and nutrient medium.

Abstract

The purpose of this work was to apply V and D cryo-plate methods for cryopreservation of Vaccinium corymbosum ʻToroʼ, Fragaria × ananassa ʻCleryʼ, and Amelanchier alnifolia (Nutt.) M. Roem. and to monitor the multiplication capacity of shoots regenerated from cryopreserved explants. Shoot tips pre-cultured for 1 d at 23°C in the dark on medium containing 0.3 M sucrose were used as explants. Loading was performed in a solution containing 2 M glycerol and 0.8 M sucrose (30 min at room temperature). In the V cryo-plate, dehydration was carried out at room temperature (20 to 50 min) using the following plant vitrification solutions: original PVS2, 90% PVS2 solution, and PVS3. Regarding the D cryo-plate, dehydration was performed in closed glass containers over silica gel for 2, 2.5, or 3 h. In both protocols, rewarming was carried out in a 1.0 M sucrose solution (15 min at 25°C). Regenerated shoots were multiplied and multiplication parameters were monitored after the second subculture. Using the V cryo-plate method, the highest regrowth in highbush blueberry was obtained following 50-min treatment with all three VSs (61.7 to 80.9%). The D cryo-plate method was even more suitable with maximum regrowth of 89.4% achieved after 2.5 h of desiccation. For strawberry, 62.5% was the highest regrowth recorded using PVS3-based V cryo-plate method while 83.3% of regrowth was observed using D cryo-plate protocol. Regrowth of saskatoon reached a maximum of 50% after 50-min treatment with PVS3 while it did not exceed 40% in other treatments. By the second subculture, shoots regenerated from cryopreserved explants regained and even exceeded the multiplication capacity of shoots regenerated from non-cryopreserved explants. This study is the first to present the successful application of the V cryo-plate method in highbush blueberry, as well as the utilization of both V and D cryo-plate methods in saskatoon.

Abstract

This study describes the in vitro regeneration of ‘Haruka’ plants, a new cultivar of Japanese flowering cherry registered in 2021 by the Japanese statutory authority. As this is a double-flowered cultivar produced by inter-specific hybridization, in vitro regeneration is an effective method for large-scale propagation. To promote proliferation, apical shoots were cultured on half-strength Murashige and Skoog medium supplemented with 5 μM 6-benzylaminopurine. The highest average of 7.8 shoots per explant was obtained at 15°C in the dark. Cultures were maintained in continuous darkness for 12 wk and then transferred to lighting conditions under a 16-h photoperiod at 25°C. Subsequently, a 100% rooting rate was obtained with the application of 1 μM indole-3-butyric acid in combination with 0.1 μM naphthaleneacetic acid. Additionally, the number of roots per shoot and the maximum length of roots were significantly higher under exposure to pink light illumination provided by cold cathode fluorescent lamps emitting red- and blue-colored light at a ratio of 80% and 20%, respectively. More than 95% of the regenerated plantlets survived after ex vitro acclimatization.

Fagonia indica Burm.f. (1768) is a medicinally important plant showing diverse pharmaceutical benefits. It is renowned for its ability to biosynthesize several anticancer and anti-inflammatory metabolites. For the eco-friendly and sustainable synthesis of phytochemicals and plant biomass, a biotechnological technique, “elicitation,” is a highly effective method in various in vitro cultures. The present study includes using various concentrations of Fusarium oxysporum Schlecht. as an elicitor in callus cultures of Fagonia indica. The main goal was to achieve enhancement in biomass production and secondary metabolism. The findings demonstrated that maximum biomass production (FW: 167.42 ± 3.99 g per 100 mL; DW: 12.53 ± 1.04 g per 100 mL) was observed at 50 mg L⁻¹ of Fusarium oxysporum as compared to the control. Secondary metabolites showed immense production (phenolic content (9.68 ± 0.23 µg mg⁻¹); flavonoid content (2.814808 ± 0.11 µg mg⁻¹)) in callus cultures treated with 10 mg L⁻¹ of Fusarium oxysporum as compared with control. Moreover, the cultures possessed the highest antioxidant capacity, as determined by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+) radical cation based assay and α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging assay, ((821.51 ± 3.20 µmol TEAC per mg DW of ABTS inhibition) (91% ± 1.45 of DPPH inhibition)) at 10 mg L⁻¹ concentration of Fusarium oxysporum, and the maximum ferric ion reducing activity (219.29 ± 2.36 µmol TEAC per mg DW) was noticed at 1.0 mg L⁻¹ concentration of F. oxysporum. Fagonia indica cultures also indicated the highest percent inhibition against cyclooxygenases (COX-1: 51.93% ± 1.74 and COX-2: 40.57% ± 1.99), lipoxygenase (15-LOX: 65.72% ± 1.44), and phospholipase A2 (sPLA2: 49.29% ± 1.75), when treated with different concentrations of F. oxysporum. HPLC analyses showed a significant accumulation of pharmacologically active components in the treated samples, with kaempferol (1245.56 mg g⁻¹) and myricetin (1139.63 mg g⁻¹) as the most accumulated compounds in the cultures with 10.0 mg L⁻¹ concentration of Fusarium in contrast to the control. These findings revealed that in callus cultures of F. indica, F. oxysporum could boost biomass accumulation and secondary metabolite production.

Glycine max (L.) Merr. (soybean) is a multi-purpose crop used for both animal and human feed. It is an economically and industrially important crop. It possesses many therapeutical and nutraceutical compounds. Therefore, soybean is referred to as ‘Gold from the soil’. Conventional breeding approaches are laborious and time-consuming. Thus, alternative biotechnological methods, such as in vitro micropropagation, regeneration, and transformation, could be advantageous. Despite the efforts made in the field of soybean micropropagation for haploidy and doubled haploidy, especially androgenesis, the success rate accounts for only approximately 2%. Androgenesis in soybean is lacking primarily due to its recalcitrant nature and differences in the development of microspores within a flower. Haploids and doubled haploids (DHs) have contributed immensely to crop improvement programs. In this review, soybean androgenesis history, production of haploids, and doubled haploids have been highlighted. The factors responsible for the androgenic responses have also been discussed. Furthermore, the review will be helpful in understanding the challenges in the standardization of protocol for the production of haploids and DHs in soybean, which will eventually assist breeding and crop improvement.

Abstract

In this study, an efficient protocol was developed to obtain multiplicated, healthy sweet basil plantlets with larger leaves and a greener color by culturing them on MS medium containing 1.0 mg/l 2iP. Chitosan and zinc oxide nanoparticles (ZnO NPs) were used as abiotic elicitors to maximize the main secondary metabolites (phenolic and flavonoid compounds and essential oil) in in vitro plantlets of sweet basil. Chitosan with a concentration of 4.0 mg/l recorded the maximum value of caffeic acid (1.189 mg/g DW), and 10 mg/l of ZnO NPs recorded the maximum content of rutin (0.936 mg/g DW). The plantlets cultured with 2.0 mg/l chitosan recorded the maximum percentage of essential oil (90.63%). 8,9-Dihydro-9-formyl cycloisolongifolene was the major compound (72.44%) and was detected in the plantlets treated with 2.0 mg/l chitosan. The inhibitory activity evaluation of α-amylase revealed that the maximum inhibitory activity was found in the plantlet control and those treated with 4.0 mg/l chitosan of the phenolic and flavonoid fractions. The maximum IC₅₀ (7.36 ± 0.85) was found in the plantlet extract treated with 4.0 mg/l chitosan, followed by the plantlet control, which recorded 11.53 ± 0.86. Using AutoDuck 4.2 and PyMol software, it was revealed that rutin and caffeic acid compounds (the main active components) own hydrogen binding interactions and hydrophobic binding interactions with the active site of alpha-amylase from the porcine pancreas (PPA) enzyme with good binding energy (5.74 and 5.71 kcal/mol, respectively).

Abstract

Bergenia ligulata, commonly known as ‘Pashanbheda’ or Indian rhubarb, is a perennial herb that has been recognized for its diverse medicinal properties. The indiscriminate use of B. ligulata has brought the species to the brink of becoming threatened. This research aims to establish a robust tissue culture protocol that can be utilized for the rapid micropropagation of B. ligulata. This protocol is essential for ensuring the sustainable production of this valuable plant species and preventing the depletion of its natural populations. The study successfully demonstrated an efficient in vitro regeneration in B. ligulata, using leaf and petiole explants. The most effective combination for achieving the highest number of shoots on either explant (leaf or petiole) involved using Murashige and Skoog (MS) medium supplemented with 0.9 µM and 1.8 µM 6-benzylamino purine (BAP) with 0.5 µM 1-naphthaleneacetic acid (NAA). Moreover, multiple shoots were also produced on MS medium fortified with 8.8 µM BAP and 2.3 µM kinetin (Kn). To achieve optimal rooting, the 45-d-old shoot was carefully isolated and placed in a half-strength MS medium. PCR-based molecular analysis using inter simple sequence repeats (ISSR) confirmed the genetically clonal nature of regenerated plantlets. About 80% of the well-developed in vitro regenerated plants were acclimatized in the glasshouse, thereby showing the robustness of the developed protocol. Based on the present study, a reproducible in vitro technique was utilized to achieve direct regeneration of approximately 3597 plants from a single explant over a 1-yr period. This approach involved molecular fidelity analysis and scanning electron microscopy (SEM) analyses to ensure reliable results.

Seedless watermelons are triploid hybrids (3n) that have been growing in popularity since the 1990s. However, the high production cost and low germination rate of the hybrid seeds pose a major problem in their production. The synseed technology could make a promising alternative for seedless watermelon propagation; however, this alternative has not yet been explored. This study aimed at producing synseeds of seedless watermelon ‘Quality’ using shoot tips as the propagules after which their germination and conversion were evaluated under in vitro and ex vitro conditions. In vitro shoot multiplication with different concentrations of 6-benzylaminopurine (BAP), synseed production from BAP-multiplied shoot tips, synseed sowing in vitro using several substrates and medium supplements, and synseed sowing ex vitro using different antimicrobial agents were investigated. Results indicated that 1.0 mg L⁻¹ BAP resulted in the highest number of shoots per shoot tip explant. Synseeds produced from BAP-multiplied shoot tips achieved faster germination compared to synseeds from non-BAP-derived shoot tips, but there was no difference in terms of conversion and acclimatization survival rates. Agar showed greater conversion and acclimatization survival rates as well as root number, root length, and shoot length than the other substrates especially when indole-3-butyric acid (IBA) at 1.0 mg L⁻¹ was added to the medium. However, this study demonstrated the possibility of using vermiculite as an alternative substrate to agar. PPM at 0.5% was a better microbial agent than 100.0 mg L⁻¹ mancozeb for ex vitro synseed sowing as it achieved zero contamination and maintained the growing potential of synseeds with 65% germination, 85% survival, and 25% rooting rates.