Bud Size Research Articles

Influence of tree water potential in inducing flowering in Rhododendron arboreum in the central Himalayan region

Rise in temperature has been reported as the principal cause of variation in flowering phenology in several tree species around the globe. In this study, we hypothesized that not only temperature but also rainfall periodicity, soil moisture and the related changes of twig water potential (ψ) in winter and early spring are important drivers of bud expansion and flowering in Rhododendron arboreum in central Himalayas. To this purpose, phenological and physiological variables (flowering time, flower bud size and twig water potential) were monitored over two years in a wild population of R. arboreum (Uttarakhand, India) and related with environmental variables (rainfall, soil moisture and temperature). Results showed that a rise in twig ψ to -0.7MPa, one week after moderate winter precipitation resulted in flower bud enlargement. In both years flowering was triggered after twig ψ reached the threshold of -0.5 Mpa, though the starting date differed between years. Floral bud size was correlated positively with twig ψ (r = 0.43, df =162, p < 0.001) and soil moisture (r = 0.61, df = 71, p < 0.001), while temperature did not influence flower bud size, soil moisture and twig ψ. Flower bud size increment was related with increase in twig ψ and soil moisture. Based on our results, we concluded that water availability plays an important role in inducing flowering in R. arboreum.

Hierarchically-structured silver nanoflowers for highly conductive metallic inks with dramatically reduced filler concentration.

Silver has long been employed as an electrically conductive component, and morphology-dependent properties have been actively investigated. Here we present a novel scalable synthesis method of flower-shaped silver nanoparticles (silver nanoflowers, Ag NFs). The preferential affinity of citrate molecules on (111) surface of silver enabled spontaneous anisotropic growth of Ag NFs (bud size: 250~580 nm, single crystalline petal thickness: 9~22 nm) with high reproducibility and a high yield of >99.5%. The unique hierarchical structure resulted in coalescence of petals over 80~120 °C which was practically employed in conductive inks to construct percolation pathways among Ag NFs. The ink with only 3 wt% of Ag NFs provided two orders of magnitude greater conductivity (1.008 × 105 Scm−1), at a low curing temperature of 120 °C, compared with the silver nanoparticle ink with a much higher silver concentration (50 wt%). This extraordinary property may provide an excellent opportunity for Ag NFs for practical applications in printable and flexible electronics.

AP1 transcription factors are required to maintain the peripheral taste system.

The sense of taste is used by organisms to achieve the optimal nutritional requirement and avoid potentially toxic compounds. In the oral cavity, taste receptor cells are grouped together in taste buds that are present in specialized taste papillae in the tongue. Taste receptor cells are the cells that detect chemicals in potential food items and transmit that information to gustatory nerves that convey the taste information to the brain. As taste cells are in contact with the external environment, they can be damaged and are routinely replaced throughout an organism's lifetime to maintain functionality. However, this taste cell turnover loses efficiency over time resulting in a reduction in taste ability. Currently, very little is known about the mechanisms that regulate the renewal and maintenance of taste cells. We therefore performed RNA-sequencing analysis on isolated taste cells from 2 and 6-month-old mice to determine how alterations in the taste cell-transcriptome regulate taste cell maintenance and function in adults. We found that the activator protein-1 (AP1) transcription factors (c-Fos, Fosb and c-Jun) and genes associated with this pathway were significantly downregulated in taste cells by 6 months and further declined at 12 months. We generated conditional c-Fos-knockout mice to target K14-expressing cells, including differentiating taste cells. c-Fos deletion caused a severe perturbation in taste bud structure and resulted in a significant reduction in the taste bud size. c-Fos deletion also affected taste cell turnover as evident by a decrease in proliferative marker, and upregulation of the apoptotic marker cleaved-PARP. Thus, AP1 factors are important regulators of adult taste cell renewal and their downregulation negatively impacts taste maintenance.

Optimizing protocol for efficient microspore embryogenesis and doubled haploid development in different maturity groups of cauliflower (B. oleracea var. botrytis L.) in India

Microspore culture technique is widely used in Brassicas for development of haploids and doubled haploids (DH) in breeding programmes and genetic studies. Indian cauliflower, a group of vegetable Brassica; with tolerance to higher temperature and moisture, has been selected in India for last 200 years. In India, cauliflowers are grown throughout the year in different climatic zones based on their temperature adaptation. For the first time, we report microspore embryogenesis in different maturity groups of Indian cauliflowers. The main objective of our study was to optimize protocol for efficient microspore embryogenesis in different types of cauliflower cultivated in India. We studied different factors like bud size, developmental stage of the microspores, culture density and heat shock treatments for efficient microspore embryogenesis in four different genotypes representing four different maturity groups. Among different factors, the bud size and microspore developmental stage was found to be more critical for successful microspore embryogenesis. The optimum bud size for high frequency microspore embryogenesis for the two early maturity groups (September–October maturity) was 4.0–4.5 mm, whereas, it was 4.5–5.0 mm for the cultivars grown for November onwards harvesting. These optimized bud sizes contained the highest percent of viable microspores and maximum number of microspores at late uninucleate to early binucleate stage. The microspore density of 6–8 × 104 cells per mL was found most suitable for efficient microspore embryogenesis. A post-culture incubation of microspores at 30.0 °C/32.5 °C for 24 h followed by maintenance at 25 °C was found optimum for induction of microspore embryos in different maturity group of Indian cauliflower. The flow cytometry analysis of 646 microspore derived plants revealed that more than 60 % plants were spontaneous diploids and less than 15 % were haploids. However, the genotypes from different groups showed varied response in different culture density and temperature treatments. Thus, it is necessary to optimize genotype specific protocol for efficient microspore embryogenesis before its routine application. The protocol developed in this study will facilitate doubled haploids based breeding and genetic studies of all types of cauliflower grown in India.

Effect of polyamines and natural growth substances on the growth and flowering of rose (Rosa hybrida) cv. Samurai under protected conditions

Investigations were conducted to study the effect of different polyamines and natural growth substances as a pre harvest foliar spray on greenhouse rose cv. Samurai. The study involved preharvest foliar spraying with polyamines like spermine (10 ppm) and spermidine (10 ppm); natural growth substances like enriched banana pseudostem sap (1 per cent) and cow urine (2 per cent). All the treatments improved the vegetative and flowering characters over control. However, among different treatments, foliar spray of spermine 10 ppm, followed by spermidine 10 ppm were highly significant in influencing all vegetative parameters like plant height (79 cm), number of branches per plant (3.73), stem girth (10.69 mm), number of leaves per plant (91.33), leaf area (14.68 cm2) and leaf chlorophyll content (36.96 mg/g). Further, flowering parameters like flower stalk length (61.24cm), bud length (2.70mm), flower diameter (6.50 cm), number of petals per flower (55.90) and vase life (6.63 days) were significantly maximum in plants sprayed with spermine 10ppm. The treatment of foliar spray with spermine and spermidine almost doubled the flower production and improved the flower quality in tems of bud size and vase life as compared to control.

Size of testes, ovaries, uterus and breast buds by ultrasound in healthy full-term neonates ages 0-3 days.

Hormonally sensitive organs in the neonate can change size within days of birth as circulating maternal estrogen wanes. Although several reports document the size of these organs through infancy, few focus attention on the near-birth period. Clinical and research evaluation of hormonal and genitourinary disorders would benefit from reference size standards. We describe the size of the uterus, ovaries, testes and breast buds in healthy term neonates. As part of the Infant Feeding and Early Development (IFED) study, we sonographically measured the largest diameter of these organs in sagittal, transverse and anterior-posterior planes for 194 female and 204 male newborns up to 3days old. We calculated mean, median and percentiles for longest axis length and for volume calculated from measured diameters. We evaluated size differences by laterality, gender and race and compared our observations against published values. Mean length and mean volume were as follows: uterus, 4.2cm and 10.0cm3; ovary, 1.0cm and 0.2cm3; testis, 1.1cm and 0.3cm3 (0.4cm3 Lambert volume); female breast bud, 1.2cm and 0.7cm3; male breast bud, 1.1cm and 0.6cm3. Breast buds were larger in females than males. Laterality differences were typically below the precision of clinical measurement. No significant race differences were detected. Using data from our large cohort together with published values, we provide guidelines for evaluating the size of reproductive organs within the first 3days of age. Discrepancies between our results and published values are likely attributable to technique.

Differential susceptibility and suitability of domestic and wild apple species for a florivorous weevil and its parasitoids

AbstractCrop plant domestication can change plant resistance to herbivores leading to differences in pest pressure experienced by crop plants and their wild relatives. To compare resistance to herbivores between domesticated and wild fruit trees, we quantified direct resistance and indirect resistance to a pest insect, the florivorous apple blossom weevil Anthonomus pomorum (Coleoptera: Curculionidae), for the cultivated apple Malus domestica and two wild apple species, the European crab apple M. sylvestris and the exotic M. kirghisorum. We measured weevil infestation and performance (weight, sex ratio), and weevil parasitism by parasitoid wasps for different cultivars of M. domestica and for the two wild apple species. To explain weevil and parasitoid responses to different apple species, we quantified tree characteristics including nitrogen content, size of flower buds, bark roughness, tree size, tree phenology and tree position. We found significant differences in susceptibility to weevil infestation between apple species, with lowest infestation (highest apple resistance) in M. domestica and highest infestation in M. kirghisorum. The suitability of apple species also varied significantly: weevils emerging from M. sylvestris were significantly lighter than those from M. kirghisorum. Parasitism of A. pomorum by different parasitoid species was significantly higher in M. sylvestris than in M. domestica. Infestation, weevil weight and parasitism were positively related to tree characteristics: infestation to bud nitrogen content and bark roughness, weevil size to nitrogen content and bud size, and parasitism to tree height and bud density. Our study revealed marked differences between apple species in susceptibility and suitability for the pest herbivore, but also for antagonistic parasitoids. Whereas direct resistance appeared to be higher in cultivated apple, indirect resistance via parasitoids was apparently higher in wild apple trees. Our findings suggest that wild and cultivated apple trees possess different resistance traits that may be combined to optimize resistance in commercial apple cultivars.

Flower Ontogenesis and Fruit Development of Synsepalum dulcificum

Synsepalum dulcificum from the family Sapotaceae is known as miracle fruit and is a valuable horticultural species. All plant parts are of medicinal importance whereas the fruit known as magic berry, miracle berry, or sweet berry is consumed fresh. Surprisingly, very little is known on the species in terms of flower morphology and flower development. In this study, an observation on the flower morphology and flower development of miracle fruit has been made with the aid of microscopic techniques. Miracle fruit flower requires 100 days to develop from reproductive meristem to full anthesis. The flower development can be divided into six stages based on the size and appearance of the flower bud. The fruit with persistent style developed and ripened 90 days after anthesis. Heavy fruit drop was observed at 40–60 days after anthesis which contributed to the final fruit set of average of 5.06% per plant. Through this study, miracle fruit is strongly insect pollinated and prevents self-fertilization. A study on pollination ecology is needed to identify the pollinator for miracle fruit, as this is important in manipulating fruit loading in the future.

Generation of bioengineered feather buds on a reconstructed chick skin from dissociated epithelial and mesenchymal cells.

Various kinds of in vitro culture systems of tissues and organs have been developed, and applied to understand multicellular systems during embryonic organogenesis. In the research field of feather bud development, tissue recombination assays using an intact epithelial tissue and mesenchymal tissue/cells have contributed to our understanding the mechanisms of feather bud formation and development. However, there are few methods to generate a skin and its appendages from single cells of both epithelium and mesenchyme. In this study, we have developed a bioengineering method to reconstruct an embryonic dorsal skin after completely dissociating single epithelial and mesenchymal cells from chick skin. Multiple feather buds can form on the reconstructed skin in a single row in vitro. The bioengineered feather buds develop into long feather buds by transplantation onto a chorioallantoic membrane. The bioengineered bud sizes were similar to those of native embryo. The number of bioengineered buds was increased linearly with the initial contact length of epithelial and mesenchymal cell layers where the epithelial-mesenchymal interactions occur. In addition, the bioengineered bud formation was also disturbed by the inhibition of major signaling pathways including FGF (fibroblast growth factor), Wnt/β-catenin, Notch and BMP (bone morphogenetic protein). We expect that our bioengineering technique will motivate further extensive research on multicellular developmental systems, such as the formation and sizing of cutaneous appendages, and their regulatory mechanisms.

Insulin-Like Growth Factors Are Expressed in the Taste System, but Do Not Maintain Adult Taste Buds.

Growth factors regulate cell growth and differentiation in many tissues. In the taste system, as yet unknown growth factors are produced by neurons to maintain taste buds. A number of growth factor receptors are expressed at greater levels in taste buds than in the surrounding epithelium and may be receptors for candidate factors involved in taste bud maintenance. We determined that the ligands of eight of these receptors were expressed in the E14.5 geniculate ganglion and that four of these ligands were expressed in the adult geniculate ganglion. Of these, the insulin-like growth factors (IGF1, IGF2) were expressed in the ganglion and their receptor, insulin-like growth factor receptor 1 (IGF1R), were expressed at the highest levels in taste buds. To determine whether IGF1R regulates taste bud number or structure, we conditionally eliminated IGF1R from the lingual epithelium of mice using the keratin 14 (K14) promoter (K14-Cre::Igf1rlox/lox). While K14-Cre::Igf1rlox/lox mice had significantly fewer taste buds at P30 compared with control mice (Igf1rlox/lox), this difference was not observed by P80. IGF1R removal did not affect taste bud size or cell number, and the number of phospholipase C β2- (PLCβ2) and carbonic anhydrase 4- (Car4) positive taste receptor cells did not differ between genotypes. Taste buds at the back of the tongue fungiform taste field were larger and contained more cells than those at the tongue tip, and these differences were diminished in K14-Cre::Igf1rlox/lox mice. The epithelium was thicker at the back versus the tip of the tongue, and this difference was also attenuated in K14-Cre::Igf1rlox/lox mice. We conclude that, although IGFs are expressed at high levels in the taste system, they likely play little or no role in maintaining adult taste bud structure. IGFs have a potential role in establishing the initial number of taste buds, and there may be limits on epithelial thickness in the absence of IGF1R signaling.

New observations on gametogenic development and reproductive experimental tools to support seed yield improvement in cowpea [Vigna unguiculata (L.) Walp

Key messageCowpea reproductive tools.Vigna unguiculata L. Walp. (cowpea) is recognized as a major legume food crop in Africa, but seed yields remain low in most varieties adapted to local conditions. The development of hybrid cowpea seed that could be saved after each generation, enabling significant yield increases, will require manipulation of reproductive development from a sexual to an asexual mode. To develop new technologies that could support the biotechnological manipulation of reproductive development in cowpea, we examined gametogenesis and seed formation in two transformable, African-adapted, day-length-insensitive varieties. Here, we show that these two varieties exhibit distinct morphological and phenological traits but share a common developmental sequence in terms of ovule formation and gametogenesis. We present a reproductive calendar that allows prediction of male and female gametogenesis on the basis of sporophytic parameters related to floral bud size and reproductive organ development, determining that gametogenesis occurs more rapidly in the anther than in the ovule. We also show that the mode of megagametogenesis is of the Polygonum-type and not Oenothera-type, as previously reported. Finally, we developed a whole-mount immunolocalization protocol and applied it to detect meiotic proteins in the cowpea megaspore mother cell, opening opportunities for comparing the dynamics of protein localization during male and female meiosis, as well as other reproductive events in this emerging legume model system. Electronic supplementary materialThe online version of this article (doi:10.1007/s00497-015-0273-3) contains supplementary material, which is available to authorized users.

Assessment of variability in water lily (Nymphaea spp.)

Twelve accessions of water lily (Nymphaea spp.) differing in flower colour and opening time were selected from diverse locations of the water lily growing tracts of Thrissur district. These accessions were evaluated based on the width as well as length of leaf lamina, length of notch on lamina, length of mature flower bud, circumference of mature flower bud, diameter of fully opened flower, number of petals and number of stigmatic appendages in the flower. The study revealed that there was significant variability among the accessions for all the characters evaluated. DMRT was carried out and the accessions were grouped taking diameter of fully opened flower as well as the size of mature flower bud as the decisive characters for grouping. Significant correlations were observed between flower size and leaf size as well as flower size and length of mature flower bud. Hence, leaf size can be taken as an indicator of the flower size of the accession. Selection programmes based on length of lamina will be effective in improving the population, since PCV, GCV, H2 and GG were the highest for this character.

Genetic and morphological analysis of multi-stemmed plants of tuart (Eucalyptus gomphocephala)

The tuart–banksia woodlands of the Swan Coastal Plain in Western Australia are characteristic vegetation communities of this coastal region, and Eucalyptus gomphocephala DC. (Myrtaceae; tuart) is an iconic tree of these communities. The species primarily occurs as a tall single-stemmed tree, but at the northern end of the distribution, it also occurs in a multi-stemmed form. Growth habit is frequently used as a taxonomic character in eucalypts, with many complexes having tree and mallee forms, although the genetic characterisation of growth habit in eucalypts has been limited. We investigated the genetic and morphological differentiation among populations of tree and multi-stemmed forms of tuart at the northern end of its distribution. Although the populations showed moderate levels of genetic diversity on the basis of microsatellite markers, as might be expected from populations on the periphery of the distribution, there was no evidence of genetic differentiation associated with the tree and multi-stemmed forms. Morphometric analysis showed some differences in the size of buds and fruits among the populations. Our analysis is consistent with environmentally induced variation in tuart in near-coastal populations where plants grow on poor soils and form may be affected by wind and salt exposure. This result adds to other evidence from pines and Nothofagus of environmental rather than genetic influences on growth form, particularly in stressful environments.

Anther culture ofGlycine max(Merr.): Effect of media on callus induction and organogenesis

The present study was undertaken to standardize the doubled haploid regeneration protocol for soybean. Studies on association between floral bud size and pollen developmental stage revealed that the bud size of 2.5–4 mm containing microspore at uni-nucleate stage was most responsive for callus induction and organogenesis. Effect of different media on callus induction and plant regeneration in anther culture of three genotypes, namely, JS 335, SL 958 and SL 688 were studied. Combination of 10.0 mg l−12, 4-D and 0.5 mg l−1 BAP supplemented with 90 g l−1 sucrose and 10% v/v of coconut water in B5 medium was found best for callus induction (64.11%). Murashige and Skoog medium supplemented with 2.0 mg l−1 BAP + 1.0 mg l−1 thidiazuron gave shoot bud-like structures, in contrast, B5 medium promoted only root organogenesis (3.30–10.90%). Anther derived callus was confirmed through flow cytometry and root tip cell of anther callus derived roots to be haploid through root tip staining (chromosome number n = 20).

In vitro clonal propagation of Polyalthia longifolia var. Pendula

In vitro clonal propagation of Polyalthia longifolia var. pendula was carried out using nodal segments from mature trees as explant. Bavistin (1.0%) for 30 min and Mercuric chloride (0.1%) for 1 min resulted in maximum survivability. Highest bud break (89.67%) and maximum mean numbers of shoot buds (6.33) were observed on MS + BAP (1.0 mg/l) + IBA (0.5 mg/l) and MS + BAP (3.0 mg/l) + NAA (0.5 mg/l) media respectively. Mean bud size was found maximum (24.6 mm) on ½ MS + IBA (100 mg/l) + 0.25 per cent activated charcoal medium. Maximum survivability (65.33%) and maximum bud break (90.33%) was observed in months of April-May. Alow response (2.5%) to rooting was shown in ½ MS + IBA (1.5 mg/l) medium where a single root, measuring 1.5 cm in length was formed in 150 days after culturing.

Androgenic potential and anther &lt;i&gt;in vitro&lt;/i&gt; culture of &lt;i&gt;Lagenaria siceraria&lt;/i&gt; (Molina) Standl an edible-seed cucurbit

Lagenaria siceraria Molina has a high productive potential of fruits but the seeds weight remain low. The aim of this study is to regenerate doubled haploids from androgenesis in order to create genotypes with good agronomic characteristics. For this purpose, flower buds of six accessions namely NI185, NI202, NI215, NI276, NI356 and NI271 were pre-treated at 4 °C for 0, 1, 2, 4, 6, 7 days. To assess the ability of these accessions to induce callus, the anthers excised from the collected floral buds were cultured following two standard protocols referred to as Metwally et al. and Kumar and Murthy, respectively. Histological sections were performed with buds of different sizes in order to select pollen at uni-nucleate stage, the most appropriate for the androgenesis. The histological sections showed that the pollen is at uni-nucleate stage when the bud size varied from 5 to 7 mm. No callus production was observed with the protocol of Metwally et al. whereas a significant formation of callus were expressed on medium corresponding to the protocol of Kumar and Murthy. The best rate (79%) of callus induction was obtained after one and two days of pre-treatment at 4 °C. The highest rate of callus induction was observed with the accessions NI271, NI276 and NI356. © 2015 International Formulae Group. All rights reserved. Keywords: Cucurbitaceae, anther culture, thermal shock, androgenesis, plant regeneration

Arecoline Alters Taste Bud Cell Morphology, Reduces Body Weight, and Induces Behavioral Preference Changes in Gustatory Discrimination in C57BL/6 Mice.

Arecoline, a major alkaloid in areca nuts, is involved in the pathogenesis of oral diseases. Mammalian taste buds are the structural unit for detecting taste stimuli in the oral cavity. The effects of arecoline on taste bud morphology are poorly understood. Arecoline was injected intraperitoneally (IP) into C57BL/6 mice twice daily for 1-4 weeks. After arecoline treatment, the vallate papillae were processed for electron microscopy and immunohistochemistry analysis of taste receptor proteins (T1R2, T1R3, T1R1, and T2R) and taste associated proteins (α-gustducin, PLCβ2, and SNAP25). Body weight, food intake and water consumption were recorded. A 2-bottle preference test was also performed. The results demonstrated that 1) arecoline treatment didn't change the number and size of the taste buds or taste bud cells, 2) electron microscopy revealed the change of organelles and the accumulation of autophagosomes in type II cells, 3) immunohistochemistry demonstrated a decrease of taste receptor T1R2- and T1R3-expressing cells, 4) the body weight and food intake were markedly reduced, and 5) the sweet preference behavior was reduced. We concluded that the long-term injection of arecoline alters the morphology of type II taste bud cells, retards the growth of mice, and affects discrimination competencies for sweet tastants.

Size Does Matter: Staging of Silene latifolia Floral Buds for Transcriptome Studies.

Dioecious plants in the Caryophyllaceae family are susceptible to infection by members of the anthericolous smut fungi. In our studies of the Silene latifolia/Microbotryum lychnidis-dioicae pathosystem, we were interested in characterizing the plant-pathogen interaction at the molecular level before and during teliosporogenesis. This takes place during floral bud development, and we hoped to capture the interaction by Illumina Next-Gen RNA-Sequencing. Using previous literature that documented the stages of the floral buds for S. latifolia, we examined the floral buds from plants grown and infected under growth chamber conditions, using the disserting microscope to determine the stage of floral buds based on the morphology. We compiled the information and determined the size of floral buds that correspond to the desired stages of development for tissue collection, for the purpose of RNA-sequencing. This offers a practical approach for researchers who require a large number of floral buds/tissue categorized by stages of development, ascertaining whether infected/uninfected buds are at comparable stages of development and whether this also holds true for male vs. female buds. We also document our experience in infecting the plants and some of the unusual morphologies we observed after infection.

Changes in Dormant Phase and Bud Development of ‘Fuji’ Apple Trees in the Chungju Area of Korea

Abstract: In this study, we investigated the onset and release of endo-dormancy under natural conditions by observing bud break characteristics in ‘Fuji’ apple trees using water cuttings. Through examinations of bud break rate and days to bud break, we found that the endo-dormancy of ‘Fuji’ apple tree continues for 70 d from 165 to 255 d after full bloom (DAFB), from late October to early January of the following year. In addition, within 20 d of first bud break, based on a final bud break rate of 60% or more, we able to identify the timing of the changeover from para-dormancy to endo-dormancy, and endo-dormancy to eco-dormancy. Analysis of the chilling requirement during the endo-dormancy period revealed that chilling accumulation up to 255 DAFB to release endo-dormancy amounted to 666 and 517 h based on the CH and Utah models, respectively. Observation of internal changes in the bud during endo-dormancy showed that flower bud differentiation begins from mid-July a, nd time of inflorescence of the disk flower is available to find. The flower buds subsequently developed slowl y but steadily during endo-dormancy and in the following year in February, the developmental stage of each organ had pro gressed. Moreover, the flower buds of ‘Fuji’ apples were mostly healthy during the dormancy period, but some exhibited necrosis of flower primordium, due partial cell damage from the formation of ice crystals rather than a direct effect of the low tempera ture. Flower buds were formed in both the axillary buds of bourse shoots and terminal buds of spurs, but lower bud differentiation was observed for the terminal buds of spurs at rate of about 65% of total buds, which was directly related to the bud size and shoot diameter.Additional key words:

MICROSPORE CULTURE IN ELEVEN CULTIVARS OF LOQUAT

In plant breeding, microspore embryogenesis via isolated microspore culture is increasing as a method to obtain in a single-step doubled haploids and homozygosity. In this study, isolated microspore culture of eleven cultivars of loquat (Eriobotrya japonica (Thunb.) Lindl.) has been carried out. An analysis of anthers from different flower bud sizes, has been carried out, to identify and to select the flower bud size with anthers containing the highest quantity of vacuolated microspores. After a pre-treatment of the panicles at 4°C in dark for 4 days, flower buds were sterilized and microspores were isolated and cultured in two different media (P and MB). Moreover, only the isolated microspores cultured in P Medium were subjected to two additional thermal shocks: cold shock (1 h at -20°C) and heat shock (7 days at 33°C). All the cultures were placed in a growth cabinet at 26±1°C in the dark for 30 days and later placed in the light. Samples from cultures were processed for microscopy analysis at specific times. Observations on microspore development were carried out by fluorescence and light microscopy. The sporophytic development of isolated microspores in culture has been confirmed by the presence in the cultures of binucleated with two equal-size vegetative-type nuclei that had just started their sporophytic pathway, multinucleated, pollen-derived multicellular structures, and calli. The results are an advancement towards the knowledge of pollen embryogenesis in loquat.