Anther Wall Research Articles

Aliphatic Metabolism during Anther Development Interfered by Chemical Hybridizing Agent in Wheat

ABSTRACTChemically‐interfered male sterility (CIMS) system in wheat (Triticum aestivum L.) is one of the male sterility types used for hybrid wheat production in China. The main morphological defect in male sterile line 1376‐CIMS interfered by chemical hybridizing agents (CHA) is that the tapetum seems to be abnormally degrated and pollen extine is aberrant. As a complex pollen wall in wheat, the pollen outer wall mainly contains lipidic sporopollenin. However, the mechanism for synthesizing these aliphatic precursors during pollen development has not been fully elucidated. To elucidate the relationship between aliphatic mechanism and chemically‐interfered male sterility, we report on the function of SQ‐1 (a new kind of chemical hybridizing agent) in aliphatic metabolism and its disruptive role during wheat pollen development. The observations of scanning electron microscopy revealed that anther and pollen wall formation was significantly altered in 1376‐CIMS. The contents of aliphatic compositions of anther underwent changes in 1376‐CIMS revealed by gas chromatography–mass spectrometry (GC–MS), including in particular reductions in fatty alcohols, alkanes and alkenes, and an abnormal increase in fatty acids in 1376‐CIMS. cDNA‐AFLP data revealed that an array of genes implicated in a diversity of biological pathways (180 in total, 124 up‐expression and 56 down‐expression) exhibited statistically significant expressional differences between 1376 and 1376‐CIMS. Also, a group of genes putatively involved in aliphatic transport and metabolism were significantly altered in 1376‐CIMS, indicating the disruptive role of SQ‐1 in aliphatic transport and metabolism in the formation of the anther and pollen wall. In addition to its function in promoting tapetum programmed cell death (PCD), SQ‐1 probably plays crucially perturbative roles in several basic biological processes during wheat anther development.

Anther wall and pollen development in Neotropical species-rich Miconia (Melastomataceae)

Miconia is one of the largest exclusively Neotropical genera, inserted in the tribe Miconieae. Although the monophyly of the tribe has recently been recognized, the delimitation of its genera is considered quite arbitrarily defined, mainly due to the great diversity combined with little morphological and genetic characterization of its members. Recent findings have associated this diversity with polyploidy, apomixis and hybridization, mechanisms commonly found in the group. The conventional delimitation of sections in Miconia, which has been largely based on stamen morphology, has proved to be undoubtedly artificial; nevertheless, the potential taxonomic significance of anther wall and pollen ontogenetic characters within the genera Miconia has been little explored. Hence, this study intended to fill that gap in our knowledge, by investigating the anther wall and pollen development in six closely related species of Miconia: M. albicans, M. fallax, M. latecrenata, M. paucidens, M. pepericarpa and M. stenostachya. Routine techniques for both light and electronic microscopy were used to examine anthers and pollen grains in several developmental stages. The species studied here share several character states, such as the remarkable monocotyledonous anther wall development, the persistent endothecium in the mature anthers with no cell wall thickenings, the bilocular dehiscent anther and the psilate pollen exine. This study also reports for the first time bisporangiate anthers, crystals and proteinoplasts in tapetal cells for Melastomataceae, and ultrastructural features of anther and pollen wall for Miconia.

Genesis of megaspore and microspore as well as development of female gametophyte and male gametophyte in Liriope spicata (Thunberg) Loureiro

The megasporogenesis and microsporogenesis, development of male and female gametophytes, pollen morphology and changes of anther wall structure during pollen development and ovary wall structure during ovule development in Liriope spicata (Thunberg) Loureiro of genus Liriope Loureiro in family Ruscaceae Spreng were studied using paraffin sections, scanning electron microscopy and fluorescence microscopy. The main results can be concluded as follows: 1) the cytokinesis of microsporocyte is successive; 2) the tetrads of microspores are mostly symmetrical and occasionally tetrahedral; 3) the mature pollen grain is oval or oblong, 2- or 3-celled, and has a sulcus and a rugulate–perforate ornamentation; 4) anther wall is of monocot type with secretory tapetum; 5) ovary is superior and has axial placentation and anatropous, bitegmic ovule; 6) the tetrads of megaspores are arranged most in a straight line and occasionally in T-shape; 7) the megaspore at the chalazal end develops into a functional megaspore; 8) development of embryo sac is of the polygonum type; 9) the floral morphology of both genera Liriope Loureiro and Ophiopogon Ker Gawler supports that each of them should be an independent genus.

The Rice Basic Helix-Loop-Helix Transcription Factor TDR INTERACTING PROTEIN2 Is a Central Switch in Early Anther Development.

In male reproductive development in plants, meristemoid precursor cells possessing transient, stem cell-like features undergo cell divisions and differentiation to produce the anther, the male reproductive organ. The anther contains centrally positioned microsporocytes surrounded by four distinct layers of wall: the epidermis, endothecium, middle layer, and tapetum. Here, we report that the rice (Oryza sativa) basic helix-loop-helix (bHLH) protein TDR INTERACTING PROTEIN2 (TIP2) functions as a crucial switch in the meristemoid transition and differentiation during early anther development. The tip2 mutants display undifferentiated inner three anther wall layers and abort tapetal programmed cell death, causing complete male sterility. TIP2 has two paralogs in rice, TDR and EAT1, which are key regulators of tapetal programmed cell death. We revealed that TIP2 acts upstream of TDR and EAT1 and directly regulates the expression of TDR and EAT1. In addition, TIP2 can interact with TDR, indicating a role of TIP2 in later anther development. Our findings suggest that the bHLH proteins TIP2, TDR, and EAT1 play a central role in regulating differentiation, morphogenesis, and degradation of anther somatic cell layers, highlighting the role of paralogous bHLH proteins in regulating distinct steps of plant cell-type determination.

Morpho-Anatomical Analysis of Cosmostigma racemosum (Asclepiadoideae) Flowers

<em>Cosmostigma racemosum</em> is a plant species belonging to the family Apocynaceae, the subfamily Asclepiadoideae. <em>Cosmostigma racemosum </em>is found in Nglanggeran Mountain Gunungkidul, Yogyakarta. The local name and its original distribution are not known. Information or study of <em>Cosmostigma racemosum</em> in Indonesia is not available. Comprehensive characterization of this species is important for authentication and addition of data base. Characterization was conducted by analyzing the morphology and anatomy of flower. The objectives of this study were to describe and analyze the morphology and anatomy of <em>C. racemosum</em> flowers. The method of research was based on observation method and exploration of plant systematics evidence or taxonomy evidence, including analyses and description of morphology and anatomy of flower structure and its development. The results showed that the characteristics of flower morphology are in accordance with the existing description in literatures. Characteristics of pollinia are specific characters of morphological aspect of flower. Data of anatomy of flower and its parts development are the new ones which confirm the position of <em>C.racemosum</em> as a member of the tribe Marsdenieae. The data of anatomy also show new information of the ontogeny of the important parts of flower: pollinia formation, pollinia corpusculum, anther wall, anther sac, stigma, stamen, staminal tube, stigmatic chamber, and structure of ovary in Asclepiadoideae

The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90).

Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes.

Anther and pollen development in the lodgepole pine dwarf mistletoe (Arceuthobium americanum) staminate flower

The lodgepole pine dwarf mistletoe, Arceuthobium americanum Nutt. ex Engelm., is a parasitic angiosperm that infects conifers in western Canadian forests. While production of viable pollen in anthers is critical to dwarf mistletoe reproduction, the few existing reports that examine staminate development in Arceuthobium are often incomplete or conflicting. The objective of this work was to investigate the developmental anatomy of anther and pollen of A. americanum using modern microscopy. We found that the microsporangium was toroidal from the outset and gave rise to a central peg-shaped sterile “columella” early in anther development. The endothecium was absent, the epidermis persisted as an “exothecium” fulfilling the role of the endothecium, and a primary parietal layer generated a secretory tapetum and middle layer. Thus, we suggest that a new category of anther wall development, the Arceuthobium type, be created. Microsporogenesis produced tetrahedral microspores via simultaneous cytokinesis and involved callose wall formation. Microgametogenesis resulted in round and atypical generative and vegetative nuclei. Additionally, the heterocolpate, echinate pollen grains, which were shed at the two-celled stage, were seen for the first time in their native state with environmental scanning electron microscopy. This work contributes to the understanding of A. americanum, the genus Arceuthobium, and angiosperms as a whole.

ABORTIVE POLLEN DEVELOPMENT IN RELATION TO EARLY TAPETAL DEGRADATION IN MALE STERILE MANGOSTEEN

Pollen development in the apomictic mangosteen (Garcinia mangostana L.) has not been exhaustively studied. The objective of this study was to establish the developmental sequence and to determine whether the bursting of the tetrads/ microspores at a time corresponding to microspore release was the factor that contributed to male sterility. Flower buds of various sizes were collected and investigated through combined anatomical observations, histochemical studies, and fluorescence microscopy. The developmental stages were categorized as follows; early pollen mother cell (eaPMC), late pollen mother cell (laPMC), tetrads (TD), early microspore (eaM) and late microspore (laM). Recent studies have shown that the tapetum was disintegrated at the laPMC stage resulting in a significant decrease in the number of tetrads. A scattered primexine wall was deposited around the tetrad. A few of the released microspores could be seen but they did not develop into functional pollen grains. In contrast, most tetrads could not be librated and were degraded. The FCR test revealed that the microspores were not viable. After Oil red O staining, lipid stores were detected in all parts of the anther. Accumulations of carbohydrate were rarely detected (PAS reaction) in the anther wall layers, tapetal cells, sporogenous derivatives and loculi. Male sterility is probably caused by the early degeneration of the tapetum combined with a disruption of the formation of tetrads/microspores. Further studies of the sub-cellular level using analysis by transmission electron microscopy (TEM) are still the subject of intensive research.

Pollinium development in Spiranthes sinensis (Pers.) Ames.and Cymbidium pendulum SW: a comparative study

Comparative studies on the early anther development, anther wall differentiation, pollinium development and pollen grains surface features in Spiranthes sinensis (Pers.) Ames. (a terrestrial orchid) and Cymbidium pendulum Sw. (an epiphytic orchid) using light and scanning electron microscopy were described. Anther primordium initiated as a homogenous mass of meristematic cells which developed two thecae, each with a group of archesporial cells of dense cytoplasmic contents. The sporogenous cells of the hypodermal layer formed the anther wall which was 4-5 layered in S. sinensis and 7-layered in C. pendulum. The rare feature of 2-layered endothecium in S. sinensis and a 2-layered tapetum in C. pendulum was reported. Papillate epidermis was observed in C. pendulum. A complete septum of sterile cells in S. sinensis and partial in C. pendulum was observed. Pollen grains were mono-aperturate, reticulate and shed as tetrads in S. sinensis while either monoaperturate or inaperturate, tectate pollen grains and dispersed as hard pollinia in C. pendulum. This is a see-through information for accurate classification and phylogenetic reconstructions of orchid species. DOI: http://dx.doi.org/10.3329/bjb.v42i2.18035 Bangladesh J. Bot. 42(2): 307-314, 2013 (December)

IAA-peroxidase relation in the microsporocytes and anther wall during successive stages of meiosis in Larix europaea L.

During anther meiosis in <em>Larix europaea</em> considerable variations in the level of peroxidase activity and endogenous auxin content occur both in the microsporocytes and in the anther wall. However, the IAA-peroxidase relations are different in each of these two parts of the anther. In the anther wall characterized by the occurrence of anodic isoperoxidases, the changes in peroxidae activity show a positive correlation with those in endogenous auxin content. In the microsporocytes containing almost only cathodic asoperoxidases the levels of endogenous auxin content and peroxidase activity show a reverse correlation. Thus a preponderance of isoperoxidases showing IAA-oxidase properties occur only in the microsporocytes. These results suggest the important role of the IAA=peroxidase system in the mechanism of differentiation of cells undergoing anther meiosis.

Variations of total protein content and protein synthesis during microsporogenesis in Larix europaea L.

The variations of the total protein content and protein synthesis were analyzed in successive stages of microsporogenesis in <em>Larix europaea</em> L. Pure fractions of microsporocytes containing the cells in successive phases of meiosis and fractions of the anther wall cells were used to perform the assays. It was revealed, that the protein content and the intensity of protein synthesis undergo considerable changes in the course of meiosis. It was proved also, by means of electrophoretic separation of protein fractions, that no new proteins are synthesised during differentiation of microsporocytes of larch, and that the microsporogenesis process is related rather to disappearance of some protein fractions.

少花龙葵花药发育中多糖和脂滴的组织化学研究

对少花龙葵花药发育中淀粉粒和脂滴的分布和转化过程进行组织化学研究。结果显示：在造孢细胞时期幼小花药中没有营养物质积累；在小孢子母细胞时期，花药表皮和药室内壁细胞中出现淀粉粒，同时在绒毡层细胞中出现了较多的脂滴；在四分体时期，中层细胞中出现淀粉粒，而绒毡层细胞中的脂滴减少；到小孢子早期，花药表皮细胞中除淀粉粒外还出现了脂滴；但到小孢子晚期，药壁细胞中的淀粉和脂滴都明显减少；在二胞花粉早期，花粉开始积累淀粉粒，药壁细胞中的淀粉和脂滴几乎消失；在即将开花的成熟花粉中积累了大量淀粉粒和少量脂滴作为其存储物。 The distribution of polysaccharides and lipid drops in developing anther of Solanum photeinocarpum was studied. At the sporogenous cell stage, neither starches nor lipids were in anther wall cells. At the stage of microspore mother cells, some starches appeared in epidemic cells and endothecium cells and many lipids appeared in tapetal cells. At the tetrad stage, some polysaccharides appeared in middle layer cells. After microspores were released from tetrads, both starches and lipids appeared in epidemic cells, but both materials decreased at the late microspore stage. After the microspore division, early bicellular pollen began to accumulate polysaccharides, and the starches and lipids disappeared in anther wall cells. With the pollen development, many starches and some lipids were accumulated in the vegetative cell of the nearly mature pollen.

Structure of staminate flowers, microsporogenesis, and microgametogenesis in <em>Helosis cayennensis</em> var. <em>cayennensis</em> (Balanophoraceae)

We analyzed the microgametogenesis and microsporogenesis of the male flowers of the holoparasitic Helosis cayennensis (Sw.) Spreng. var. cayennensis using optical and scanning electron microscopy. The unisexual flowers are embedded in a dense mass of uniseriate trichomes (filariae). Male flowers have a tubular 3-lobed perianth, with bilayered and non vascularized tepals. The androecium consists of three stamens with filaments and thecae connated into a synandrium. It has adnate a free central pistillode without megagametophyte. Staminal filaments, fused at their base to the perianth tube and distally free along a short section, have a single vascular bundle. The distal portion of the synandrium is formed by nine pollen sacs: six outer sacs are located laterally to each filament and three longer inner sacs. The anther wall consists of the epidermis, two parietal layers (that collapse at anther maturity), and an uninucleate secretory tapetum. There is no endothecium. During microsporogenesis, the stem cells produce tetrads of microspores by meiosis. The cytokinesis is simultaneous, forming tetrahedrally arranged tetrads. When pollen grains are in the tricellular state, the synandrium emerges from the mass of filariae, and anthers dehiscence occurs apically through longitudinal slits. In conclusion, despite the extreme reduction of flowers, the anatomic characteristics and gametophyte development of staminate flowers of H. cayennensis are perfectly normal and functional. They are thus highly similar to other genera of the holoparasitic subfamily Helosidoideae. Sterile parts of flowers and inflorescence maintain the same distinctive and aberrant features of the plant vegetative parts.

A Study of Microsporgenesis and Male Gametogenesis in <em>Camellia grijsii</em> Hamce

<em>Camellia grijsii</em> Hamce is used as a woody edible oil tree and wellknow for its commercial value in Southern China. The plants rarely set viable seeds and have little work on the reproduction biology. In order to verify whether there was any obstacle of male reproduction in the <em>C. grijsii</em>, microsporgenesis and male game to genesis in <em>C. grijsii</em> have been evaluated by paraffin section technique. The results are showed that the development of the anther wall belonged to a basic type and consisted of epidermis, endothecium, middle layers and tapetum. The tapetum conformed to the glandular type. Cytokinesis during meiosis of the microspore mother cell was simultaneous type. A majority of the microspores were arranged in tetrahedral tetrads. The mature pollen grains were 2-cell type and had three germ pores. Anthers were dehiscent and pollen grains shed on the early-February. Based our results, we did not find the abnormal male flower in the <em>C. grijsii</em>, suggesting that male gametes were fertile and male sterility was not the major cause of the low seed set in the <em>C. grijsii</em>.

Induction of Quercus ilex L. haploid and doubled-haploid embryos from anther cultures by temperature-stress

Abstract This paper describes a method to obtain haploid and doubled-haploid (DH) embryos using anther cultures of holm oak (Quercus ilex L.). The production of haploids and DH through gametic embryogenesis provides an attractive biotechnological tool for developing homozygous lines from heterozygous parents, which is important in breeding programs, as well as in genetic studies. As a consequence, protocols to produce homozygous plants have a significant impact on forest tree improvement. Anthers were subjected to different temperature treatments for embryo induction: a cold pre-treatment (4°C) from 3 to 7 days was carried out at the beginning, followed by a heat shock (33°C) from 2 to 5 days. Most anthers responding to these stress treatments contained vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in holm-oak microspores. In all cases, embryos grew from the interior of the anthers, breaking through the degenerating anther walls. Under these conditions, embryo formation occurred in 31 anthers between 46 and 95 days after culture initiation. Embryo analysis performed with flow-cytometry and DNA-microsatellite markers showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages. This is, to our knowledge, the first published report on gametic embryogenesis in holm oak.

Embryology ofAbeliophyllum(Oleaceae) and its phylogenetic relationships

Abeliophyllum, a monotypic endemic genus of Oleaceae, resembles Forsythia in various morphological characters, but its phylogenetic position is disputed and no embryological study of the genus has been carried out. We investigated more than 40 embryological characters of Abeliophyllum, compared them with previous information on Oleaceae, and discusses its phylogenetic relationships. Abeliophyllum is similar to other genera of Oleaceae in many embryological features, having some distinct features such as the mode of anther wall formation, formation of a nucellar cap, and formation of obturator and hypostase. The basic type of anther wall development and formation of a nucellar cap have not previously been reported in Oleaceae. In addition, differentiation of the obturator and formation of hypostase are not reported in the previously investigated genera of the family. Compared with close relatives, the seed coat structure of Abeliophyllum resembles Forsythia more than Fontanesia and supports existing molecular data which place Abeliophyllum as the sister group of Forsythia.

Identification of the tapetum/microspore-specific promoter of the pathogenesis-related 10 gene and its regulation in the anther of Lilium longiflorum

A tapetum/microspore-specific pathogenesis-related (PR) 10 gene was previously identified in lily (Lilium longiflorum Thunb.) anthers. In situ hybridization and RNA blot analysis indicated that the lily PR10 genes are expressed specifically and differentially in the tapetum of the anther wall and in microspores during anther development. The accumulation of PR10 transcripts was exogenously induced by gibberellic acid (GA) and was suppressed by ethylene. Studies using inhibitors of GA and ethylene revealed that the lily PR10 is modulated by an antagonistic interaction between GA and ethylene. The treatment of norbornadien, an ethylene inhibitor, caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole, an inhibitor of GA biosynthesis, arrested tapetal development to a status close to that of control. The expression of the lily PR10g promoter in transgenic Arabidopsis was determined using the β-glucuronidase (GUS) reporter gene indicated that the decisive fragment required for anther specificity is located −1183bp to −880bp upstream of the transcription start site. The PR10gPro::barnase transgenic lines exhibited complete male sterility because of the disruption of the tapetum and the deformation of microspore/pollen. The anther specificity of lily PR10 highlights the importance of the tapetum/microspore-specific PR10g promoter for future biotechnological and agricultural applications.

A Morphological and Histological Characterization of Male Flower in Chestnut (&lt;em&gt;Castanea&lt;/em&gt;) Cultivar 'Yanshanzaofeng'

Chinese chestnut (Castanea mollissima Blume.) is a widely distributed fruit tree and well known for its ecological and economic value. In order to evaluate obstacles to male reproductive in the C. mollissima, a morphological and histological characterization of male flower of chestnut cultivar 'Yanshanzaofeng' were examined by paraffin section technique and scanning electron microscopy. The results showed that male catkins with floral primordia were formed in the buds of one-year olds shoots in later April. Later, a protoderm, ground meristem and a procambium had differentiated in young anthers. Each young anther soon developed to four microsporangia. The anther wall layers developed completely by mid-May and consisted of one-cell-layered epidermis, one-cell-layered endothecium, two or three middle layers and one-cell-layered tapetum. The tapetum was of glandular type. Microspore mother cells underwent meiosis through simultaneous cytokinesis in later May and gave rise to tetrads of microspores, which were tetrahedrally arranged. Mature pollens contained two cells with three germ pores. Anthers were dehiscent and pollen grains shed by early June. Based our results, we did not find the abnormal male flower in the C. molissma cv 'yanshanzaofeng', indicating that male gametes were fertile and thus was considered as pollenizers.

Anther wall development, placentation, sporogenesis and gametogenesis in Smilax davidiana A.DC: A contribution to the embryology of Smilax

Embryological characters can be used to address taxonomic relationships and complement molecular phylogenetics and are of special value at the genus level. However, embryological information is fragmentary in Smilax and completely unknown in Smilax davidiana, a Chinese species. Anther wall development, placentation, sporogenesis and gametogenesis of S. davidiana are characterized here. The anther is bisporangiate, anther wall formation is of the Dicotyledonous type, both epidermis and endothecium develop fibrous thickenings, and the tapetum is secretory and of dual origin. Cytokinesis in the microsporocyte meiosis is successive, the microspore tetrad is tetragonal, and mature pollen is two-celled. The ovary is mostly trilocular with an axile placentation (a small fraction of the ovaries are unilocular with parietal placentation), the ovule is anatropous, bitegmic and crassinucellate, with embryo sac development of the Polygonum type. This study documents for the first time the embryological characters of S. davidiana in detail and contributes much to the embryology of Smilax.

Tapetum and middle layer control male fertility in Actinidia deliciosa

Background and AimsDioecism characterizes many crop species of economic value, including kiwifruit (Actinidia deliciosa). Kiwifruit male sterility occurs at the microspore stage. The cell walls of the microspores and the pollen of the male-sterile and male-fertile flowers, respectively, differ in glucose and galactose levels. In numerous plants, pollen formation involves normal functioning and degeneration timing of the tapetum, with calcium and carbohydrates provided by the tapetum essential for male fertility. The aim of this study was to determine whether the anther wall controls male fertility in kiwifruit, providing calcium and carbohydrates to the microspores.MethodsThe events occurring in the anther wall and microspores of male-fertile and male-sterile anthers were investigated by analyses of light microscopy, epifluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and transmission electron microscopy coupled with electron spectroscopy. The possibility that male sterility was related to anther tissue malfunctioning with regard to calcium/glucose/galactose provision to the microspores was also investigated by in vitro anther culture.Key ResultsBoth tapetum and the middle layer showed secretory activity and both degenerated by programmed cell death (PCD), but PCD was later in male-sterile than in male-fertile anthers. Calcium accumulated in cell walls of the middle layer and tapetum and in the exine of microspores and pollen, reaching higher levels in anther wall tissues and dead microspores of male-sterile anthers. A specific supply of glucose and calcium induced normal pollen formation in in vitro-cultured anthers of the male-sterile genotype.ConclusionsThe results show that male sterility in kiwifruit is induced by anther wall tissues through prolonged secretory activity caused by a delay in PCD, in the middle layer in particular. In vitro culture results support the sporophytic control of male fertility in kiwifruit and open the way to applications to overcome dioecism and optimize kiwifruit production.