Characterization of mode of reproduction in Commiphora wightii [(Arnot) Bhandari] reveals novel pollen–pistil interaction and occurrence of obligate sexual female plants

Guggul [Commiphora wightii (Arnot) Bhandari], a polygamous woody tree valued for its medicinal oleoresin gum rich in guggulsterone, is reported to reproduce via sporophytic apomixis. Details about its natural diversity, and mode and extent of sexual reproduction are, however, scanty. Therefore, a comprehensive investigation of guggul reproduction was made employing histology, controlled pollination, flow cytometry and RAPD analyses of progeny to assess the occurrence and extent of sexual reproduction. We report the discovery of obligate sexual female plants of guggul through these studies. Also, we document a unique pollen–pistil incompatibility that prevents all but one pollen tube growth into the style to effect fertilization. Consequently, obligate sexual female plants produced single-seeded fruit although each flower contains four ovules. In apomictic plants bearing more than one seed per fruit, at most only one seed was of sexual origin. Further, flow cytometric analysis conclusively demonstrated that endosperm development occurs either autonomously or following triple fusion. Autonomous endosperm development was invariably associated with endoreduplication, a unique feature of apomixis in guggul. Despite predominance of apomixis, a low frequency of sexual reproduction was found to persist in apomictic plants yielding new genetic variation. RAPD analysis clearly distinguished accessions and was useful in identifying sexual progenies. The implications of the novel pollen–pistil interaction on establishment and spread of apomixis in guggul are discussed. The study has not only revealed novel features of guggul reproduction but also opened new opportunities for molecular genetic analysis of sporophytic apomixis and breeding improvement of guggul.     

Pollen-pistil Interaction in a Non-pseudogamous Apomict,Commiphora wightii

Structural and cytochemical details of the pistil and the interactionof pollen and pistil were studied in a non-pseudogamous apomict,Commiphorawightii.The anthers in the male and bisexual flowers producefunctional pollen grains. The stigma is of the wet and papillatetype. The style is typically solid with two strands of transmittingtissue that traverse the entire length of the style. There isa marked reduction in the area occupied by the transmittingtissue from the stigma to the base of the style. The cells ofthe transmitting tissue are isodiametric in transverse as wellas longitudinal section and do not form longitudinal files ofelongated cells as reported for other taxa. Proteins could notbe localized in the intercellular matrix. Although pollen grainsgerminate on the stigma, pollen tubes do not grow beyond theproximal one third of the style. Changed orientation of thecells of the transmitting tissue and absence of proteins inthe intercellular matrix could account for the failure of thepistil to support pollen growth.Copyright 1998 Annals of BotanyCompany Guggul, pollen-pistil interaction, non-pseudogamous apomict,Commiphora wightii, transmitting tissue     

花椒和野花椒的无融合生殖

花椒与野花椒的胚囊发育类型属蓼型,成熟胚囊的卵器退化。花椒无雄花,不发生双受精,自发形成胚乳并产生珠心胚。野花椒虽有正常花粉,人工授粉后能萌发,但在花粉管长入胚囊之前卵器已解体,中央细胞中已形成胚乳游离核,因此也不发生双受精,由珠心细胞自发形成胚。这种现象是花椒和野花椒在长期进化过程中形成的一种十分特化的适应。     

Sporophytic apomixis in polyploid Anemopaegma species (Bignoniaceae) from central Brazil

Apomixis and polyploidy have been important in the evolution of the angiosperms, and sporophytic apomixis has been associated with polyembryony and polyploidy in tropical floras. We studied the occurrence of polyembryony in populations of tetraploid Anemopaegma acutifolium, A. arvense and A. glaucum from the Brazilian cerrados, and histological features of sexual and apomictic processes were investigated in A. acutifolium. All populations and species were polyembryonic (68.9–98.4% of seeds). Normal double fertilization occurred in most ovules, with exceptions being that 3% of ovules were penetrated but not fertilized and in 4% of ovules both synergids were penetrated. The penetration of both synergids suggests a continuous attraction of pollen tubes and polyspermy. Adventitious embryo precursor cells (AEPs) arose from nucellar and integumental cells of the ovule in pollinated and unpollinated A. acutifolium, indicating sporophytic apomixis. However, further embryo and endosperm development required pollination and fertilization. This pseudogamy also allows concurrent sexual embryo development. Similar polyembryony rates and polyploidy indicated that A. arvense and A. glaucum are also apomictic, forming an agamic complex similar to that observed for some species of confamilial, but not closely related Handroanthus. The co‐occurrence of apomixis and polyploidy in different groups of Bignoniaceae indicates homoplasious origin of these agamic complexes. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 77–91.     

An improved method of DNA purification from secondary metabolites rich medicinal plants using certain chaotropic agents

The presented work describes good quality DNA isolation method from mature leaves of some medicinally important plant species, viz. Asparagus racemosus, Withania somnifera, Abrus precatorius, Commiphora wightii and Carissa carandas. These plants hold immense medicinal values due to presence of certain secondary metabolites like polyphenols, terpenes, flavonoids, alkaloids, gums, resins, etc. Although these metabolites are accountable for important medicinal properties and authorize these plants to precedence over others, the same compounds disappoint the researcher while isolating high quality DNA. To overcome this problem, we propose a simple method in which DNA is adroitly bounded to diatomaceous earth in a solution of different chaotropic agent and alienated from intrusive compounds. Presented method affirms that secondary products, along with polysaccharides and proteins, can be perceptibly reduced by using silica matrix along with chaotropic agents. The described method is fast, simple and highly reliable for the isolation of DNA from obstinate plant species.     

Formation of Multiple or Adventive Embryos in Epidendrum nocturnum Jacq (Orchidaceae)

Under greenhouse conditions, Epidendrum nocturnum Jacq. plantsproduce fruits by both self-fertilization and cleistogamy. Althoughadapted to these reproductive processes the species respondalso to cross-pollination. Seeds without embryos and with oneembryo are usual but occasionally seeds with two, three or fourembryos are produced. Multiple embryos are formed by polyembryonyand apomixis. Epidendrum nocturnum, self-pollination, cleistogamy, apomixis, polyembryony, supernumerary embryos     

Storage-protein Deposition in the Developing Rice Caryopsis in Relation to the Transport Tissues

The developing caryopsis of rice (Oryza sativa L.) was examinedhistologically at successive stages of grain-filling in orderto identify the factors which determine the distribution ofstorage protein in the endosperm, and which terminate the depositionof endosperm protein. The storage protein was deposited at theperiphery of the endosperm, and this distribution was apparentlycaused by the radial pattern of cell development in the endosperm,and by the proximity of the peripheral endosperm cells to thenucellar epidermis. The nucellar epidermis directly surroundsthe endosperm and functions as the pathway for amino acid transportto the endosperm. During the later stages of caryopsis developmentthe nucellar epidermis became compressed by being ‘sandwiched’between the expanding endosperm and the rigid hull (the tightlylocked palea and lemma) which encloses the caryopsis. It isproposed that this compression of the nucellar epidermis blocksthe supply of amino acids to the endosperm and thereby terminatesthe deposition of storage protein in the rice grain. Oryza sativa, rice, caryopsis (development), endosperm, grain filling, nucellar epidermis, storage protein     

AFLP markers closely linked to a major gene essential for nucellar embryony (apomixis<Emphasis Type="Italic">)</Emphasis> in <Emphasis Type="Italic">Citrus maxima</Emphasis> × <Emphasis Type="Italic">Poncirus trifoliata</Emphasis>

Some citrus varieties express a form of apomixis termed nucellar embryony in which the adventive embryos develop from nucellus tissue surrounding the embryo sac. This trait results in many seeds containing multiple embryos (polyembryony). Inheritance of the frequency of polyembryony was studied in 88 progeny from a cross of Citrus maxima (monoembryonic) × Poncirus trifoliata (polyembryonic). The frequency of polyembryonic seed produced by each progeny was determined by scoring 100–500 seeds for the number of seedlings to emerge from each seed. Two groups of eight individuals from each extreme of the population were chosen for bulked segregant analysis with amplified fragment length polymorphism markers amplified with 256 primer combinations. Candidate markers identified in the bulks as linked to the trait were tested on the 32 individuals used to create the bulks and then on the remaining plants in the population. Five candidate markers tightly linked to polyembryony in P. trifoliata were identified. Specific marker alleles were present in nearly all progeny that produced polyembryonic seed, and alternate alleles were present in nearly all progeny that produced only monoembryonic seed. The region defined by these markers very likely contains a gene that is essential for the production of polyembryonic seeds by apomixis, but also shows segregation distortion. The proportion of polyembryonic seeds varied widely among the hybrid progeny, probably due to other genes. Scoring 119 progeny of a P. trifoliata selfed population for the closely linked markers and the proportion of polyembryonic seeds confirmed close linkage between these markers and polyembryony.     

The best of both worlds: Apomixis and sexuality co-occur in species of Microlicia,Melastomataceae

Apomixis, the asexual formation of embryos and seeds, occurs in approximately 18% of angiosperm families. Melastomataceae exhibits a remarkable number of apomictic species, distributed among different tribes. This mode of reproduction has been elucidated in Miconieae, but remains unclarified for other groups, such as Microlicieae. Although apomixis has been previously described for Microlicieae species, the cytological basis for this phenomenon is entirely unknown in this group. Thus, populations of Microlicia fasciculata and M. polystemma were used in order to (a) investigate the presence of autonomous apomixis; (b) verify if this mode of reproduction leads to polyembryony; and (c) investigate whether apomixis may occur in parallel with the sexual process. We tested these species for autonomous fruit set and polyembryony, and pollen viability, and analyzed pollen tube growth. Anatomical techniques were used to elucidate the micro- and megasporogenesis and gametogenesis. The species showed autonomous fruit and seed formation and exhibited polyembryony. Apospory and adventitious embryony were the developmental mechanisms of apomixis in M. fasciculata and M. polystemma, respectively. Both species exhibited low pollen viability. However, some viable pollen, reduced embryo sac formation, natural pollination and pollen tube growth enable sexual reproduction and characterize these species as facultative apomicts. The independence of pollinators for fruit set, uniparental reproduction and the possibility of sexual reproduction, confer reproductive assurance and flexibility, bringing together advantages of sexual and asexual reproduction. In this sense, apomixis may have played an important role in the evolution and diversification of Microlicia, a widely distributed genus in the Brazilian Cerrado.     

Development and Structure of Primary Secretory Ducts in the Stem of Commiphora wightii (Burseraceae)

Development of gum-resin ducts, sites of resin synthesis inthe epithelial cells and elimination of resin from the protoplasthave been studied in the stem of Commiphora wightii. Formationof an intercellular space amongst a group of densely stainedprocambial cells signals the initiation of a duct. It widensby anticlinal divisions of the epithelial cells and also bytheir further separation along the radial walls. The numerousplastids with varying morphological shapes have an electrondense matrix. Starch granules present in the amyloplasts showevidences of exocorrosion. Mitochondria in the epithelial cellsof developing and mature ducts have well developed, swollencristae. Osmiophilic material originates in association withthe golgi-derived vesicles at its maturing face. It is alsoobserved in close association with plastids, mitochondria andvacuoles, thereby plausibly involving them in the process ofresin formation. The resinous material is eliminated from thecytoplasm by vesicles enveloped by plasmalemma prior to theirdischarge into the apoplast. Myelin-like multilamellate structuresobserved along the inner tangential wall may aid in the secretionof resin across the wall. Commiphora wightii, primary secretory ducts, epithelial cells, ultrastructure, gum-resin secretion     

Embryology of Early Abortion Due to Limited Maternal Resources in Pisum sativum L.

In most flowering plants, many embryos are aborted early intheir development due to limited maternal resources. The kin-conflictinterpretation of plant embryology predicts these abortionsshould be under maternal control. In a study of the abortionprocess in Pisum sativum, we found the first visible indicationof abortion was formation of a weak hypostase. Callose was locallydeposited around the chalazal endosperm haustorium, and ligninalong the outer cell walls of the remnant nucellar tissue. Thenucellus was compressed by proliferating adjacent inner integumentalcells. The endosperm haustorium's cytoplasm was forced backinto the embryo sac cavity. With suppression of haustorial activitythe endosperm nuclei gradually enlarged followed by enlargementof the embryo and suspensor nuclei. Finally, nuclei and cytoplasm throughout the endosperm and embryolost stainability and broke down. Four successive stages wererecognized in seed abortion. In seeds developing to maturity,no hypostase was developed and the haustorium continued to digestboth the remnant nucellus and the proliferated inner integumentalcells. These observations are consistent with the kin-conflicthypothesis. Pisum sativum, garden pea, ovule abortion, histology, hypostase, kin-conflict hypothesis     

Evolution of polyembryony: Consequences to the fitness of mother and offspring

Polyembryony, referring here to situations where a nucellar embryo is formed along with the zygotic embryo, has different consequences for the fitness of the maternal parent and offspring. We have developed genetic and inclusive fitness models to derive the conditions that permit the evolution of polyembryony under maternal and offspring control. We have also derived expressions for the optimal allocation (evolutionarily stable strategy, ESS) of resources between zygotic and nucellar embryos. It is seen that (i) Polyembryony can evolve more easily under maternal control than under that of either the offspring or the ‘selfish’ endosperm. Under maternal regulation, evolution of polyembryony can occur for any clutch size. Under offspring control polyembryony is more likely to evolve for high clutch sizes, and is unlikely for low clutch sizes (<3). This conflict between mother and offspring decreases with increase in clutch size and favours the evolution of polyembryony at high clutch sizes, (ii) Polyembryony can evolve for values of “x” (the power of the function relating fitness to seed resource) greater than 0.5758; the possibility of its occurrence increases with “x”, indicating that a more efficient conversion of resource into fitness favours polyembryony. (iii) Under both maternal parent and offspring control, the evolution of polyembryony becomes increasingly unlikely as the level of inbreeding increases, (iv) The proportion of resources allocated to the nucellar embryo at ESS is always higher than that which maximizes the rate of spread of the allele against a non-polyembryonic allele.     

Partially Fertile Line with Apospory Obtained from Tissue Culture of Male Sterile Plant of Sorghum (Sorghum bicolor L. Moench)

A partial restoration of male fertility has been revealed inthree among 20 regenerants from callus cultures obtained fromthe panicle fragments of the sorghum plant with cytoplasmicmale sterility. Induced fertility is retained under self-pollinationfor eight generations. The pollen fertility level in every generationvaries in different plants (0-70%) and depends on the environmentalfactors, rather than on plant genotype. A high positive correlation(r = 0·90; P < 0·01) has been revealed betweenthe pollen fertility level and the total rain precipitationduring the microgametophytogenesis. Cytoembryological investigationof this line has shown the presence of structures similar toaposporous embryo sacs (ESs), which have developed in the ovulesalong with the sexual ESs. The ovules with aposporous formationshave been observed almost in all of the studied plants in severalgenerations, however, their frequency varied (2-53%). Parthenogeneticproembryos and endosperm nuclei without signs of pollen tubepenetration have been found in some of such ESs. The natureof the instability observed in a number of generations in maleand female generative structures is discussed.Copyright 1995,1999 Academic Press Cytoplasmic male sterility, fertile revertants, apomixis, somaclonal variability, Sorghum bicolor L. (Moench)     

Structural Differentiation of the Nucellar Epidermis in the Caryopsis of Rice (Oryza sativa)

In the developing caryopsis of rice (Oryza sativa L.) the nucellarepidermis forms a uniseriate layer through which assimilatesare transported to the endosperm. An anatomical study demonstratedthat the nucellar epidermal cells are fusiform in shape andare hexagonally packed. The anticlinal walls of the nucellarepidermis are characterized by ribs of wall-thickening whichare orientated radially with respect to the caryopsis. The wall-thickeningsappear to be cellulosic primary walls, as indicated by theirstaining with Calcofluor and periodic acid-Schiff's reagent.It is proposed that the geometry of the nucellar cells and theribs of wall-thickening are structural adaptations to resistthe compressional force which is placed on the nucellar epidermisduring the latter stages of grain filling. Oryza sativa, rice, caryopsis, grain filling, nucellar epidermis, wall-thickening     

Phenomenon of polyembryony. Genetic heterogeneity of seeds

Different concepts of polyembryony and genetic heterogeneity of seeds in flower plants have been reviewed. Different types, ways, and forms of plant reproduction appeared in the course of evolution as a consequences of the attached mode of life and autotrophy. This is ascribed to totipotency, “stemminess” of plant cells, and presence of constantly functioning meristems, which determined to a great extent the system of plant safety. There are two ways of formation of a new individual: sexual process → gamospermy involving meiosis and gamete fusion and asexual process → agamospermy without meiosis and gamete fusion and two types of reproduction: seed and vegetative. Both processes may take place simultaneously in one seed, as a result of which many embryos of different origins are formed: uniparental and biparental inheritance. Traditionally, this phenomenon is called polyembryony. It comprises embryoidogeny (a new category of vegetative reproduction): formation of somatic embryos (= embryoids) in the flower, seed, and on vegetative organs. Genetic heterogeneity is one of the most important characteristics of seeds, which is based on different phenomena, such as embryogeny, embryoidogeny, and gametophytic and sporophytic apomixis. When describing two types of polyembryony, sporophytic (nucellar, integumental, cleavage) and gametophytic (synergidal, antipodal), a great attention is paid to characterization of initial cells of the sexual and adventive embryos. A new concept of apogamety is developed from new positions (totipotency and “stemminess”), which is based on different genesis of cells of the egg and antipodal systems. Five possible pathways of formation of the adventive embryos have been proposed from cells of the egg apparatus. Specific features of the formation of adventive embryos in the case of gametophytic apomixis, such as androgenesis and semigamy, are discussed. Morphogenesis of the sexual and adventive embryos proceeds in the mother organism and is determined by the origin and formation of their initials, types of ovule and embryo sac, and specific features of developmental biology. This determines parallelism in their development. The main difference consists in the way of reproduction: heterophasic and homophasic. The phenomenon of polyembryony and genetic heterogeneity of seeds is essential for development of the theory of reproduction and applied research related to seed productivity of plants.     

Apomixis in <Emphasis Type="Italic">Eulaliopsis binata</Emphasis>: characterization of reproductive mode and endosperm development

Apomixis represents an alteration of classical sexual plant reproduction to produce seeds with essentially clonal embryos, stimulating wide interest from biologists and plant breeders for its ability to fix heterosis. Eulaliopsis binata (Poaceae), is identified here as a new apomictic species. Embryological investigation indicates that the developmental pattern of embryo sac formation in E. binata represents gametophytic apospory, the embryo originating from an unreduced cell, without fertilization and the mode of endosperm development was autonomous. Sexual embryo sacs were found with a frequency of 1–4% depending on the biotype. The DNA content of nuclei (C-value) in mature seeds was screened by flow cytometry (FCSS) and demonstrated that the endosperm was derived autonomously without fertilization and the three biotypes of E. binata showed varying degrees of apomixis. The Wide-leaf type showed obligate apomixis whereas the Slender-leaf and the Red-haulm type displayed facultative apomixis. In addition, adventitious embryos were observed on the wall of ovary, integument and nucellus cells, indicating that E. binata produces embryos via a mixture of apospory and adventitious embryony.     

Apomixis in Taraxacum paludosum (section Palustria, Asteraceae): Recombinations of apomixis elements in inter-sectional crosses

Recent studies have shown independent control of apomixis elements (restitution/diplospory, parthenogenesis and autonomous endosperm) in Taraxacum sect. Ruderalia. We studied inheritance of apomixis elements in the section Palustria using the crosses between various sections used as mother plants and apomictic T. paludosum (sect. Palustria) as pollen donor. Non-apomictic plants prevailed in F₁ progeny, and a high incidence of sterility was observed. Triploid non-apomictic F₁ hybrids were backcrossed with diploids (sects. Ruderalia, Palustria) and tetraploids (sects. Palustria, Piesis), and produced various types of progeny. These F₁ hybrids were classified into three types depending on the occurrence of parthenogenesis along with restitution, and the occurrence of various progeny in particular crosses (i.e. within the same mother plant) was observed. The results indicate the independent genetic control of all apomixis elements in T. paludosum, and recombinations during a restitutional megasporogenesis in hybrids.     

Biogenic synthesis of gold nanoparticles using Commiphora wightii and their cytotoxic effects on breast cancer cell line (MCF-7)

The current study aimed at developing gold nanoparticles (AuNPs) using the aqueous extract of the medicinal plant Commiphora wightii. The phytosynthesized gold nanoparticles (Cw@AuNPs) were evaluated for their anticancer activity against MCF-7 breast cancer cell model. The formation of AuNPs by Commiphora wightii leaf extract was confirmed by UV–vis spectra where their surface plasmon resonance was found at 533 nm. Further characterization of Cw@AuNPs was done by transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive X-ray (EDX) analysis, and fourier-transform infrared spectroscopy (FTIR) analysis. In vitro anticancer potential of thus obtained AuNPs was evaluated against MCF-7 and where the IC₅₀ was found to be 66.11 μg/mL Further, apoptotic studies were carried out using ethidium bromide dual staining, DNA fragmentation, comet assay, and flow cytometry studies. Results revealed that Cw@AuNPs at higher concentration significantly increased the apoptotic cells when compared to control cells. Cell cycle analysis of MCF-7 cells confirmed the cell cycle arrest at G2/M phase. These results demonstrate that the biosynthesized Cw@AuNPs appear to be promising for therapeutical applications against breast cancer.     

Polyembryony in Melastomataceae from Brazilian Cerrado: multiple embryos in a small world

Polyembryony has been commonly associated with apomixis in the angiosperms and seems to be more common than expected, even in biomes where sexual reproduction processes are predominant. Recent studies in Cerrado, the Neotropical savannas of Central Brazil, showed high frequencies of apomixis and polyembryony and indicated these processes as reproductive and evolutionary alternatives for plants in these areas. In this sense, we investigated the occurrence of polyembryony and its relationships with ecological (season and type of dispersal, ploidy, species distribution and breeding system) and taxonomic (tribe) factors in the Melastomataceae, a mostly tropical family already known for its high frequency of apomixis and very common in Cerrado. We collected seeds from 69 populations of 53 species, which were sown in germination chambers. After seed germination, the presence and number of seedlings per seed were evaluated as a method to estimate polyembryony. We encountered 18 species (33.96%) with polyembryony (more than one seedling, or gemellar seedlings, originated per seed) concentrated in species of the tribe Miconieae (64%) and Microlicieae (16.67%), but absent in Melastomeae. Monoembryony was present only in sexual species, while all apomictic species were polyembryonic. In Miconia, the polyembryony was correlated with polyploidy, and monoembryony with diploid species. Polyembryony was more common among species with wide distribution in the Cerrado region, which indicates that the presence of gemellar seedlings is important for establishment and survival of the group in the Cerrado biome.