Horse chestnut pollen quality

Pollen quality of horse chestnut, expressed as pollen productivity, viability and germination was studied. Anthers of horse chestnut genotypes had pollen production from 3.66 to 5.06 x 10 3 pollen grains per anther, depending of genotype. Also, pollen of horse chestnut Ah1-Ah4 genotypes showed different viability (from 56 to 68%), after staining with fluorescein diacetate. Pollen germination of Ah1-Ah4 genotypes varied from 50-66% on basic medium. Inclusion of polyethylene glycol-PEG from 10%, 15% and 20% v/w increased pollen germination. The best results were achieved on medium with the largest PEG concentration. On these medium 76-91% pollen grains were germinated, depending of genotype. The best pollen quality, for all tested parameters, had genotype Ah2. Knowledge about morphology, production, viability, in vitro germination, tube growth as well as pollen: ovule ratio can be of great importance for future pollen biology studies.Genetika (2017), 49(1): 105-11

Variability and bimodal distribution of size in uninuclear microspores of Aesculus flava Marshall

Projekat ministarstva br. 14302

Comparative analysis of microspore size variability in the genus Aesculus (Hippocastanaceae)

Pollen size varies extensively among angiosperm species and partially reflects evolutionary adaptation of each species to the pollination and fertilization environment. Size of uninuclear microspores in Aesculus parviflora was analyzed and compared with the size of microspores in Aesculus hippocastanum, Aesculus carnea, and Aesculus flava. The microspores came from closed flower buds of different size (3, 4, and 5 mm) isolated from lower (female flowers), middle (bisexual flowers), and upper (male flowers) segments of inflorescences. Aesculus parviflora had smaller microspores than Aesculus carnea and Aesculus flava, but larger microspores than Aesculus hippocastanum. All analyzed microspores showed bimodal distribution in all investigated species of the genus Aesculus.Projekat ministarstva br. 14302

Pure Camphor and a Thujone‐Camphor Mixture as Eco‐Friendly Antifeedants against Larvae and Adults of the Colorado Potato Beetle

The Colorado potato beetle (CPB) is a serious pest of economically important Solanaceae species. The use of essential oil compounds in pest management has been proposed as an alternative to harmful chemical insecticides that disturb human health and ecosystem functioning. We examined the antifeedant activity of three concentrations (0.125%, 0.25% and 0.5%) of pure camphor and a thujone-camphor mixture against 3rd instar larvae and adults. Their efficacy was evaluated according to the degree of leaf damage and avoidance of treated leaves by the CPB. Treatment of potato leaves significantly reduced leaf damage compared to the control. Leaf protection increased at higher concentrations of the examined compounds. Camphor was more effective against larvae and the thujone-camphor mixture was more effective against adults. Additionally, adults moved faster towards the control leaf disc in the two-choice olfactometer assay if an alternative disc was treated with a thujone-camphor mixture, whereas larvae responded similarly to the two potential repellents. However, after contact with the leaf disc treated with the highest compound concentration, the larvae escaped faster from the thujone-camphor mixture than from pure camphor. In conclusion, both examined compounds are promising eco-friendly antifeedants, but their efficacy depends on the developmental stage of the beetle, compound type and applied concentration

Rooting and preventing shoot-tip necrosis of in vitro cultured horse chestnut shoots

Efficient bud regeneration was achieved from germinating horse chestnut (Aesculus hippocastanum L.) somatic embryos cultivated on 1-10 μM benzyladenine (BA). Adventitious buds were detached from the mother tissue and used to establish permanent shoot cultures on 0-20 μM BA. Secondary buds were regenerated from the shoot base of the explants. Bud multiplication was very poor (1.9) and shoot-tip necrosis was very high (100%) on plant growth regulator (PGR)-free medium. The highest multiplication was achieved on 5 and 10 μM BA (16.8 and 18.7, respectively), with no shoot-tip necrosis, while hyperhydration was rather frequent on shoots cultivated on BA above 5 μM. Individual shoots were elongated on medium with 1 μM BA and 500 mg/l polyvinylpyrrolidone (PVP MW40 000) for 4 weeks. However, it was necessary to reduce BA level below 1 μM for shoot rooting and that caused mass shoot-tip necrosis. As classical rooting methods failed, the basal part of each elongated shoot was first wounded by cutting with a sterile blade and then dipped into a 0, 5 or 10 mM indole-3-butyric acid solution for 1 min and cultivated on solid half-strength MS PGR-free medium with 0.02% activated charcoal for 2-3 weeks. To prevent shoot tip necrosis during this phase, a BA solution was applied directly on apical meristem. Shoot-tip necrosis was completely eliminated by weekly application of 10 μl of 1 μM BA. As soon as the root initials were observed, the shoots were transferred to MS medium supplemented with 500 mg/l PVP and 5 μM BA. The frequency of rooting was 23%, and further optimisation of root-inducing phase is needed

Somatic embryogenesis from stamen filaments of Aesculus flava Sol. and peroxidase activity during the transition from friable to embryogenic callus

Species of the genus Aesculus are among the most attractive ornamental woody plants. Conventional propagation methods of these species are either inefficient (stem cuttings) or unsuitable for clonal propagation (seeds). The aim of the present study was to develop an efficient protocol for clonal propagation of elite specimens of yellow buckeye (Aesculus flava) by somatic embryogenesis. For this purpose, stamen filaments of yellow buckeye were cultivated on media supplemented with 1, 5 or 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D) combined with 0, 1, 5 or 10 μM 6-furfurylaminopurine (Kin), either under light or dark conditions, for 8 weeks, and then subcultivated on plant growth regulator (PGR)-free medium with 400 mg/l of glutamine. The highest somatic embryo (SE) initiation rates were achieved for the explants cultivated in darkness on medium containing 1 μM 2,4-D + 10 μM Kin during callus induction (CI) phase. Embryogenic calli (EC) were initiated from friable calli, starting from the 7th week of culture initiation, while SEs appeared two weeks later, following a week of subcultivation of the explants on PGR-free medium. EC and SEs were observed only in the explants grown in darkness during CI phase. Minimal duration of CI phase and darkness necessary for SE induction was four weeks, while the highest embryogenic response was achieved when each lasted for 8–10 weeks. Obtained SEs were efficiently multiplied on medium supplemented with 0.05 μM 2,4-D + 5 μM Kin by recurrent somatic embryogenesis. SEs at globular stage of development exhibited the highest capacity for secondary SE regeneration. High germination and conversion rates were attained in cotyledonary-stage SEs cultivated on medium with 0.05 μM 2,4-D + 5 μM Kin, but this phase needs to be further optimised, since the obtained plants failed to acclimatize to greenhouse conditions. During the transition of calli from friable to embryogenic state, total peroxidase (POX) activity significantly decreased, indicating their involvement in the acquisition of embryogenic capacity. The presented protocol is suitable for clonal propagation and genetic transformation of this ornamental species, and POX activity may be used as a marker for SE initiation

Genetic modification of horse chestnut as a tool for the ornamental traits improvement

Reduced plant size, modified habitus and changes in the flowering time may be interesting and economically important in the breeding of ornamentals. These traits could be achieved by genetic transformation with the rol genes of Agrobacterium rhizogenes. Transformed plants, more or less, exhibit traits typical for the hairy root syndrome, including the shortening of internodes abolished apical dominance, increased axilary branching, more compact habitus, changes in leaf and flower morphology, and flowering time shift. Horse chestnut was transformed by using the agropine A. rhizogenes strain A4GUS, with the uidA reporter-gene inserted in the TL-DNA. Plantlets were regenerated from the acquired hairy root lines via the process of somatic embryogenesis, and, to a lesser extent, organogenesis. Transformed plantlets displayed the characteristic hairy root syndrome at varying degrees. Plants of all lines were shorter than control ones, although branching did not increase. The line 36 had almost normal leaf morphology, while lines 43 and 31 had narrow leaves. Lines 39 and 47 displayed the most severe phenotype stunted growth, very narrow dark-green leaves and short petioles. It seems that at least line 36 may be interesting, for example, for the bonsai plants production.Redukovana veličina biljke, modifikovani habitus i promene u vremenu cvetanja mogu biti zanimljive i ekonomski značajne u oplemenjivanju ornamentalnih vrsta. Ove osobine se mogu postići genetičkom transformacijom ml genima Agrobacterium tiiizogenes. Transfoimisane biljke, u manjoj ili većoj meri pokazuju osobine tipične za "hairy root" sindrom: skraćene internodije ukinutu apikalnu dominaciju, izraženo bočno grananje, kompaktniji habitus izmenjenu morfologiju listova i cvetova i pomereno vreme cvetanja. Divlji kesten je transfonnisan pomoću agropinskog soja A. rhizogenes A4GUS, sa uidA reporter genom insertovanim u TL-DNK. Iz dobijenih linija transformisanih korenova su regenerisane biljčice procesom somatske embriogeneze i, u manjoj meri procesom organogeneze. Transformisane biljčice su u različitom stepenu pokazivale karakterističan "hairy root" sindrom. Biljčice svih transfonnisanih linija su bile kraće od netransformisanih, mada grananje nije bilo povećano. Linija 36 je imala skoro normalnu morfologiju lista, dok su linije 43 i 31 imale uzane listove. Kod linija 39 i 47 sindrom je bio najizraženiji; biljčice su bile zakržljale, sa vrlo uzanim tamno-zelenim listovima i kratkim petiolama. Čini se da bi bar linija 36 mogla biti zanimljiva, npr. za produkciju bonsai-biljaka.Projekat ministarstva br. 157

Comparative Study of Antioxidant Status in Androgenic Embryos of Aesculus hippocastanum and Aesculus flava

In vivo (leaves and seed embryos) and in vitro (androgenic embryos) antioxidant scavenging activity of Aesculus hippocastanum and Aesculus flava medical plants was examined. Here we report antioxidant enzyme activities of superoxide dismutase, catalase, guaiacol peroxidase and glutathione peroxidase, reduced glutathione quantity, flavonoids, soluble protein contents, quantities of malondialdehyde, and (OH)-O-center dot radical presence in the investigated plant samples. Total antioxidant capacity of all the samples of A. hippocastanum and A. flava was determined using FRAP, DPPH, and NO center dot radical scavenger capacity. The leaves of A. flava collected from the botanical garden exhibited stronger antioxidant activity (higher activities of SOD, and higher quantities of GSH, TSH, TPC, and scavenging abilities of DPPH and NO center dot, and higher FRAP values and lowest quantities of (OH)-O-center dot and MDA) than in vitro obtained cultures. However, the leaves of A. flava showed higher antioxidant activity than the leaves of A. hippocastanum, and therefore they have a stronger tolerance of oxidative stress. Androgenic embryos of both species had low amount of antioxidants due to controlled in vitro environmental conditions (T, photoperiod, humidity, nutritive factors, and pathogen-free). Our results confirmed that we found optimal in vitro conditions for producing androgenic embryos of both Aesculus species. Also, we assume that horse chestnut androgenic embryos can be used as an alternative source for large-scale aescin production.Ministry of Education, Science, and Technology Development of Serbia {[}III43002, 173015

Somatic Embryogenesis in Spinach—A Review

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