Globalization has drastically reduced the number of autochthon apple cultivars in the Serbian market and most of them have nearly disappeared; however, some of these cultivars, such as Petrovača, ...Budimka, Kolačara Pozna, and Kožara, have extraordinary quality, good pomological characteristics, and pest and disease resistance. The present study was conducted to develop a protocol for the storage of pollen for further use in the conservation and breeding of these cultivars. Viability and germination of the mature pollen were tested
in vitro
, at four storage temperatures (20, 4, −20, and −80°C), right after harvest or 1, 2, 3, 4, 5, and 6 months after storage. Differences in fresh pollen viability and germination between cultivars were statistically significant and ranged from 60 to 88% and 59 to 98%, respectively. Fresh pollen of cv. Budimka showed the highest viability and germination in comparison with other cultivars, especially cv. Kožara. Pollen viability and germination decreased over the storage period, and it was the lowest after 6 months of storage at room temperature in all tested cultivars. Storage at 4°C prolonged the pollen viability and germinability of 1–5 fold, depending on the cultivar and treatment duration; however, the pollen longevity of all cultivars was significantly extended when stored at −20 or −80°C. After 6 months, pollen of cv. Budimka stored at −20 and −80°C showed 14–15 fold higher germination rates in relation to pollen storage at room temperature for the same period. The results of the present study suggest that the pollen of these apple cultivars could be efficiently maintained at −20°C and could be further used for breeding purposes, e.g., for crossings between cultivars that flower at different times of the year.
High variability in somatic embryo (SE)-forming capacity has previously been observed in several spinach cultivars. Such variability frequently accounted for more variation in embryogenic response of ...the explants than the factor being investigated. Hence, the variability in embryogenic capacity was examined in the present study at both the population and the single-seedling level, using seeds of spinach cultivar Matador obtained from nine European seed companies. Seed population obtained from Slovenia (Sl) was superior to others, with the highest regeneration frequency (100%) and the highest mean SE number (14.4). A total of 82% of these seedlings had 80-100% of regenerating explants, while in populations with intermediate embryogenic capacity approximately 40% of seedlings had 20-60% of regenerating explants. The explants from the majority of seedlings (52-100%) in the least responsive populations were irresponsive. Furthermore, the explants from Sl seedlings regenerated from 10-20 (43.5%) up to > 20 (27.6%) SEs on average, while the explants from the majority of seedlings belonging to other populations regenerated 1-10 SEs. The present study strongly indicates that the variability of plant material must not be overlooked, because choosing more responsive individuals for one treatment and less responsive ones for another may lead to misinterpretation of the data.
A spinach-supplemented diet exerts numerous health benefits, but high levels of oxalic acid and nitrate can cause medical problems, so their levels should be reduced, while the levels of vitamins and ...phytochemicals could be further increased by breeding. Conventional spinach breeding is limited by the very complex sex determination. However, these limitations could be circumvented in synergy with a biotechnological approach. Accordingly, tissue culture techniques allow rapid and efficient clonal propagation of selected valuable genotypes, and somatic embryogenesis has been recognized as a superior process for clonal propagation because somatic embryos resemble zygotic embryos and therefore can spontaneously develop into complete plants. Since spinach has been considered recalcitrant to in vitro regeneration for decades, a deeper insight into the mechanisms underlying somatic embryogenesis is important for a better understanding and further improvement of the efficiency of this process. In this review, a comprehensive overview of the major factors affecting somatic embryogenesis in spinach is presented and discussed, with particular emphasis on the synergistic effects of α-naphthaleneacetic acid, gibberellic acid, light, and the intrinsic predisposition of individual seedlings to somatic embryogenesis, as well as the expression of genes encoding key enzymes involved in the maintenance of gibberellin homeostasis and the levels of endogenous gibberellins.
Three hairy root clones of
Hypericum perforatum
(HR 2, HR 15 and HR 27) transformed with
Agrobacterium rhizogenes
A4M70GUS and their corresponding regenerated shoot culture clones (HRRS) were ...compared for differences in growth, production of phenolic compounds, antioxidant and antimicrobial activities. Transgenic clones were selected on the basis of morphological evaluation, genetic and molecular analyses. The clone HR 2 had the highest biomass accumulation, while HR 27 showed the highest shoot regeneration potential. The total phenolics and flavan-3-ols were enhanced in all tested transgenic cultures, while total flavonoids and hypericins were augmented in HRRS clones compared to non-transformed shoots. The HRRS clones produced substantial amounts of chlorogenic acid and 3-
p
-coumaroylquinic acid. Regarding the flavonoids, they produced significant contents of luteolin hexoside (HRRS 2), quercitrin and quercetin (HRRS 15) and isoquercetin (HRRS 27), while HR 2 and 15 accumulated 4-
O
-methylkaempferol-
O
-hexoside and quercetin 6-
C
-glucoside, respectively. The HR 15 was promising for the production of catechin and procyanidin derivatives and together with its HRRS clone exhibited a high potential for hyperforin and adhyperforin production. All identified naphtodianthrones were confirmed in HRRS 2 and 15 clones. Among xanthones, mangiferin was found as the major compound in HRRS, while trihydroxy-1-metoxy-
C
-prenyl xanthone was dominant in HR clones. Antimicrobial activity of transgenic cultures revealed that HRRS 15 strongly inhibited the growth of
Bacillus cereus, Micrococcus flavus, Pseudomonas aeruginosa
and
Escherichia coli
. Altogether,
H. perforatum
HR and HRRS cultures could be proposed as promising experimental systems for enhanced production of phenolic compounds with antioxidant and antibacterial properties.
Over the last years, cytokinin deficiency has been studied in a variety of plant species, using transgenic expression of cytokinin oxidase/dehydrogenase genes (
CKX
). In tobacco and
Arabidopsis ...thaliana
, overexpression of
A. thaliana CKX
genes caused changes in plant morphology known as the "cytokinin deficiency syndrome", including stunted shoot growth, decreased apical dominance, and enhanced root growth. For a valid comparison with the previously obtained in vitro grown
AtCKX2
-transgenic potato lines, we report here on the construction of transgenic lines of the cultivar Désirée constitutively expressing the gene
AtCKX1
under control of the CaMV 35S promoter. Three lines expressing the transgene in both shoot and root tissues were used for analysis. Analyses of CKX activity of tissue extracts, cytokinin levels, morphology, and tuberization parameters revealed similarities as well as differences from the
AtCKX2
-transgenic plants, including lower levels of CKX activity, and more pronounced morphological effects of cytokinin deficiency. One of the three
AtCKX1
-overexpressing potato lines was able to start forming tubers before 30 days of growth in long day conditions (16 h/8 h photoperiod) as has been previously observed in some
AtCKX2
overexpressors. The extension of the experimental period to 120 days of growth in vitro showed that the other two
AtCKX1
-overexpressing lines showed impaired tuberization in terms of the number of tubers per plant, and tuber size. We conclude that cytokinin deficiency has distinct effects on tuber induction and tuber initiation/growth, possibly mediated by distinct signaling mechanisms.
Gibberellins (GAs) play a pivotal role in the induction of somatic embryogenesis from in vitro root apices of spinach plants. With the aim to understand the role of GAs in this process and to improve ...somatic embryo (SE) regeneration efficiency, the impact of light and GAs on SE initiation from the in vitro root apices was studied. The root sections were isolated from in vitro-grown SE-derived plants and placed on medium containing 20 µM α-naphthaleneacetic acid (NAA) and 0–10 µM GA
3
or GA
1
, and cultivated under light conditions or in darkness. The most efficient SE regeneration response (100% regenerating SEs and 40.73 SEs per root apices) was achieved only in the presence of both light and GAs, with GA
3
always exhibiting much stronger effect than GA
1
. Considering that light enhances GAs biosynthesis and the necessity of GAs for SE initiation, the expression levels of genes encoding the key enzymes involved in the final steps of GAs synthesis (
SoGA20-ox1
and
SoGA3-ox1
) and deactivation (
SoGA2-ox1
,
SoGA2-ox2
and
SoGA2-ox3
) were analyzed. Light enhanced the expression of all five
GA-ox
genes, while exogenously supplied NAA + GA
3
provoked downregulation of
SoGA20-ox1
and
SoGA3-ox1
and upregulation of
SoGA2ox-2
and
SoGA2ox-3
expression. The expression of
SoGA2ox-1
only slightly decreased. The results indicated the capability of isolated spinach roots to perceive the light and autonomously produce GAs. The expression levels of genes encoding key enzymes involved in GA biosynthesis suggest that lower levels of GAs favor SE initiation.
Key message
Light and gibberellins (GA) synergistically promote somatic embryogenesis in spinach. Expression levels of genes encoding key enzymes for GA metabolism suggest that lower levels of GAs may favor somatic embryogenesis.
Species of the genus
are very attractive woody ornamentals. Their organs contain numerous health-promoting phytochemicals. The most valuable of them-aescin-is used in commercial preparations for the ...treatment of venous insufficiency. The industrial source of aescin is horse chestnut seeds because the zygotic embryos are the main site of its accumulation. Horse chestnut somatic and zygotic embryos contain similar amount of aescin, hence somatic embryos could be exploited as an alternative source of aescin. Somatic embryogenesis, androgenesis and de novo shoot organogenesis were successfully achieved in several
species, as well as secondary somatic embryogenesis and shoot organogenesis, which enables mass production of embryos and shoots. In addition, an efficient method for cryopreservation of embryogenic tissue was established, assuring constant availability of the plant material. The developed methods are suitable for clonal propagation of elite specimens selected as the best aescin producers, the most attractive ornamentals or plants resistant to pests and diseases. These methods are also useful for molecular breeding purposes. Thus, in this review, the medicinal uses and a comprehensive survey of in vitro propagation methods established for
species, as well as the feasibility of in vitro production of aescin, are presented and discussed.
Flax seeds stored in unfavorable conditions were exposed to static magnetic fields (SMFs) of 50–350 mT for 20–120 min to overcome low germination and quality. Seed germination increased slightly with ...increasing strength of SMF and duration of treatment. Seed germination from 89% to 100% was achieved in SMF treatments of 150 mT (120 min), 200 mT (80–120 mT), 250 mT (60–120 min), 300 mT (40–120 min), and 350 mT (40–120 min). In these treatments, germination was 2.78- to 3.12-fold higher than in the control after 10 days. Treatments with 350 mT for 100 and 120 min showed the best results in germination (100%), as well as a 26.81-fold increase in vigor I, 28.69-fold increase in vigor II, 1-fold increase in chlorophyll a, 0.84-fold in chlorophyll b, 0.46-fold increase in carotenoid content, and 2.63-fold increase in catalase activity compared to the control after 10 days. Also, SMF treatment of 350 mT (20–120 min) reduced cell leakage and electrical conductivity by 1-fold compared to the control. SMF is a healthy, biologically safe, and environmentally friendly treatment and can be a tool for overcoming problems of low germination and quality of seeds stored under unfavorable conditions.
Kohlrabi (Brassica oleracea var. gongylodes) is an important vegetable crop that is able to undergo shoot regeneration in culture from intact seedlings in a single-step regeneration process, using ...cytokinin as the only plant growth regulator. In this work, we present the expression profiles of seven organogenesis-related genes over the time course of shoot regeneration from intact seedlings of kohlrabi cv. Vienna Purple on shoot regeneration media containing trans-zeatin, cis-zeatin, benzyl adenine or thidiazuron. Two auxin transporter genes – PIN3 and PIN4, a cytokinin response regulator – ARR5, two shoot apical meristem-related transcription factors – CUC1 and RGD3, and two cell cycle-related genes – CDKB2;1 and CYCB2;4 – displayed bimodal expression patterns on most cytokinin-containing media when their expression levels were normalized against control plants grown on hormone-free media. The first expression peak corresponded to direct upregulation by cytokinin from the growth media, and the second one reflected transcriptional events related to callus formation and/or acquisition of organogenic competence, corresponding to the shoot regeneration phases that have already been characterized in Arabidopsis thaliana. We demonstrate that the genes involved in the two-step shoot regeneration of Arabidopsis display their expected expression profiles during the single-step shoot regeneration of its close phylogenetic relative kohlrabi confirming the universality of their roles in the distinct phases of the regeneration process in Brassicaceae. The results presented here represent a first step towards genetic characterization of the morphogenetic processes in this important crop species.
Heat stress substantially reduces crop productivity worldwide, and will become more severe due to global warming. Identification of proteins involved in heat stress response may help develop ...varieties for heat tolerance. Eukaryotic elongation factor 1A (eEF1A) is a cytosolic, multifunctional protein that plays a central role in the elongation phase of translation. Some of the non-canonical eEF1A activities might be important in developing plant heat-stress tolerance. In this study, we investigated effects of heat stress (HS) on eEF1A expression at the protein level in potato, a highly heat vulnerable crop. Our results from both the controlled environment and the field have shown that potato eEF1A is a heat-inducible protein of 49.2-kDa with multiple isoforms (5–8). Increase in eEF1A abundance under HS can be mainly attributed to 2–3 basic polypeptides/isoforms. A significant correlation between eEF1A abundance and the potato productivity in the field was observed in two extremely hot years 2011 and 2012. Genomic Southern blot analysis indicated the existence of multiple genes encoding eEF1A in potato. Identification, isolation and utilization of heat-inducible eEF1A genes might be helpful for the development of the heat-tolerant varieties.
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