Induction of nucellar embryogenesis in nucellar tissue of Citrus aurantifolia for their clonal multiplication

Citrus aurantifolia (lime) has been selected as explant for nucellar embryogenesis. Nucellus is a non-vascularized tissue being true-to-type same as mother plant, meristematic cells have no plasmodesmata connection, no virus can pass through nucellus, thus it seems to be a good material for production of virus freeplantlet.Putrescine at 0.25 or 0.5 mg1-1 and anapthaleneacetic acid at 0.10 mg1-1 supplemented to nutrient formulation were most effective in alleviating cotyledonary proliferation and fasciation while promoting embryo-to-embryo proliferation producing numerous whitish globular embryos were formed. For further development of globular embryos to well-differentiated cotyledonary embryos, additional presence of 2-isopentenyladenine at concentrations of 0.10 or 0.25 mg 1-1 was essential, contrary to incorporation of 0.10 or 0.25 mg 1-1 6benzylaminopurine, which promoted excessive proliferation of cotyledonary structures and their fasciation while zeatin at the same concentrations produced intermediate response. In the optimum treatment containing 0.25 mg l-1 putrescine, 0.10 mg 1-1 isopentenyladenine, 0.10 mg 1-1 indole-3-acetic acid and 100 mg l-1 malt extract, an average 10 well-developed embryos per culture were formed, besides some abnormal cotyledonary structures. Well-developed embryos measuring ca. 2 cm. in length (leaving the root) germinated 100% into plantlets, during 60 days, in the additional presence of amino acid supplement comprising, 5 mg 1-1 each of L-arginine, L-asparagine, L-histidine, L-cysteine, L-lysine and 10 mg l-1 L-glutamine. Such plantlets nurtured in a different medium attained a height of ca. 4 cm in 45 days before they were taken out for ex vitro growth. There was 100% transplant success and the plants grew normally.

Plantlet regeneration via nucellar embryogenesis in Citrus jambhiri

To produce true-to-type and rapid multiplication micropropagation technique was utilized to the nucellar tissue with ovular halves of C. jambhiri. Nucellar tissues were cultured in a modified MS medium supplemented with different concentrations of 2ip viz. 0.25,0.50 mg l-1 alone and in combination of 0.50 mg l-1 NAA. Initiation of cell division and differentiation of proembryogenic tissue became apparent in first 80 days.These proembryos developed into embryos through subculturing in fresh medium. Low concentration of 0.25 2ip was found more suitable for the development of embryo producing large number of fully developed embryo in comparision to the embryo produced in the high concentration of 2ip (0.50 mg l-1) and in combination of 0.25 2ip and 0.50 mg l-1 NAA. Normally developed embryos in 0.25 2ip showed best germination in the fresh medium supplemented with 0.25 mg l-1 IAA, 100 ME mg l-1 and 5mg mg l-1 amino acids within 30 days as compared to other treatments. These germinated embryos were utilized for producing disease free saplings after hardening and nurturing in laboratory conditions. The disease free saplings thus produced can be used to establish new Citrus orchards within short time.

H3.1 Eviction Marks Female Germline Precursors in Arabidopsis

In flowering plants, germline precursors are differentiated from somatic cells. The female germline precursor of Arabidopsis thaliana is located in the internal (nucellar) tissue of the ovule, and is known as the Megaspore Mother Cell (MMC). MMC differentiation in Arabidopsis occurs when a cell in the subepidermal layer of the nucellar apex enters the meiotic program. Increasing evidence has demonstrated that MMC specification is a plastic process where the number and developmental outcome of MMCs are variable. During its differentiation, the MMC displays specific chromatin hallmarks that distinguish it from other cells within the primordium. To date, these signatures have been only analyzed at developmental stages where the MMC is morphologically conspicuous, and their role in reproductive fate acquisition remains to be elucidated. Here, we show that the histone 3 variant H3.1 HISTONE THREE RELATED 13 (HTR13) can be evicted in multiple subepidermal cells of the nucellus, but that H3.1 eviction persists only in the MMC. This pattern is established very early in ovule development and is reminiscent of the specific eviction of H3.1 that marks cell cycle exit in other somatic cell types, such as the root quiescent center (QC) of Arabidopsis. Our findings suggest that cell cycle progression in the subepidermal region of the ovule apex is modified very early in development and is associated with plasticity of reproductive fate acquisition.

Timing of Reproductive Organs Maturity in Proterandrous Malva sylvestris L.

Flower development of protandrous species Malva sylvestris L. was divided into 12 stages, as revealed by applying histological and scanning electron microscope techniques. Flower development started with the conversion of apical meristem into floral meristem. Initiation of male organ primordia started before that of female organ. Five rounded structures called corolla/androecium units differentiated from floral meristem, on which stamen primordia emerged. When pollen mother cells exist in the pollen sacs, initiation of carpels starts from floral meristem. Concurrent with the termination of meiosis in pollen mother cells, ten loculed ovary comed in view. Simultaneously with the occurrence of vacuoleted pollen grains, megaspore mother cell becomes visible in the nucellar tissue. Concominant with the existence of two celled-pollen grains, the style was formed and ovule becomes anatropous. When pollen grains are shed in male phase, ten branched stigma appeared on the upper part of the style, but receptive surface was not fully formed to accept pollen grains. Female phase is characterized by the opening of stigmatic branches with papillate receptive surface. Timing of reproductive organs maturity overlapped for a while, thus, the reproductive stragey of M. sylvestris was incomplete proterandry.

Pollen tube growth through nucellus into embryo sac of Spinacia - an ultrastructural investigation

The micropylar parts of nucellus and embryo sac were studied in relation to pollen tube growth and its entrance into the embryo sac. The initially homogeneous walls of the cells of the conductive nucellar tissue disintegrate at the middle lamellae region. Pollen tubes pierce the nucellar cuticle and continue their growth into the nucellus intercellularly. Subsequently they can follow various pathways to reach the FA of the degenerated synergid. The penetration into this synergid, and the discharge of the tube contents are described and discussed.

Structural Analysis of Reproductive Development in Pistillate Flowers of Laurus nobilis L.

In Laurus nobilis L. (Lauraceae) the development of female flowers (pistillate), between floral meristem differentiation and fruit formation was inspected through histological sections and SEM techniques. The reproductive development of the female flower starts when the apical meristem converts into a floral meristem. Four tepals, four stamens and a carpel are developed from the floral meristem in turn. Filaments emerge however, anther development is arrested, and stamens become nonfunctional staminodes. The stigma is of the dry type. The solid style being short and thick consists of an epidermis, a cortex, a vascular bundle and a core of transmitting tissue composed of elongated cells. In the style a funnel-shaped zone extending from within the stigma to the stylar base is visible. The presence of high amounts of sugars and lipid substances within and around the vascular bundles are identified by histochemical techniques. The ovary contains an anatropous, bitegmic and crassinucellate ovule. Starch grains are present throughout the development of nucellar tissue. The chalazal region of nuclear endosperm forms a short haustorium. Endosperm does not exist in mature seed; the cotyledons are piled with considerably large starch grains. Idioblasts are observed in all stages of development.

Embryology of the dioecious Australian endemic Lomandra longifolia (Lomandraceae)

Microsporogenesis, embryogeny and endosperm development of Lomandra longifolia Labill. are described in detail. The formation of the anther wall is the basic type composed of four cell layers, namely an epidermis, an endothecium, one middle layer and a tapetum. The tapetum layer has glandular, uninucleate cells. Successive cytokinesis follows meiosis, subsequently forming a tetrahedral tetrad of microspores. The ovule in each carpel is hemitropous, crassinucellate and bitegmic, with the micropyle formed by the inner integument. The archesporial cell divides periclinally to form the primary parietal and primary sporogenous cells. The sporogenous cell functions as the megaspore mother cell, whereas the parietal cell divides to give rise to two parietal layers. The mature megagametophyte, which has enlarged synergids and antipodals, is of the Polygonum type, with the normal complement of seven cells and eight nuclei. Nucellar tissue in the mature ovule consists of enlarged dermal cells and irregular subdermal cells surrounding a central strand of markedly smaller cells. Endosperm development is of the nuclear type. Embryo development is of the Graminad type, characterised by oblique zygotic and early pro-embryonic divisions.

Infection of Cranberry Flowers by Monilinia oxycocci and Evaluation of Cultivars for Resistance to Cottonball

Infection of cranberry flowers by conidia of Monilinia oxycocci, the cottonball pathogen, was investigated using a squash-mount histological method. Conidia germinated on anthers, nectaries, petals, and stigmata, but not styles. The stigma was the only flower part penetrated by the fungus, but no specialized infection structures were noted. Both fungal and pollen germ tubes grew through the stylar canal and made contact with ovules and nucellar tissue by 72 h after inoculation and pollination. Cottonball incidence was greatest when stigmata were inoculated; the low level of cottonball that resulted from inoculation of other flower parts and in noninoculated flowers was attributed to contamination of stigmata. In greenhouse tests, cottonball incidence was 25, 28, 31, and 38% for cvs. Searles, Pilgrim, Ben Lear, and Stevens, respectively, and was greater for M. oxycocci isolate 593 than isolate 591. We conclude that the stigma is the sole floral infection court for conidia of M. oxycocci and that the most popular cranberry cultivars in Wisconsin do not differ in inherent resistance to cottonball. The relevance of these findings to the long-term management of cottonball is discussed.

Fruit and Seed Structure of Thryptomene calycina (Myrtaceae)

At anthesis the receptacle of flowers of T. calycina (Lindl.) Stapf is enclosed by a layer of small cells with a thick cuticle. The hypodermis consists mostly of large oil-containing cells interspersed with much smaller cells. The tissue surrounding the ovary consists mostly of spongy parenchyma. The micropyle of the ovule is formed only by the inner integument which is double-layered. The embryo sac at anthesis is typical of the Myrtaceae, and appears to follow the monosporic polygonum type, with considerable nucellar tissue surrounding it. In a mature fruit the parenchyma is compressed into a thin band surrounding the seed. The integuments form a two-layered seed coat within the fruit. The aborted ovule is displaced below and to one side of the developing seed. In fruit weathered for several years there is an accumulation of phenolic material giving the fruit a black appearance. The two layers of the seed coat within freshly shed fruit lie closely together and stain strongly with the lipid stain auramine O. In fruit weathered for at least 2 years the two layers of the seed coat become separated for at least part of their length and staining from auramine O is less intense. The breakdown in seed dormancy in weathered fruits is probably due to a less complete barrier to water uptake provided by the separation of the two layers. This would increase lateral and radial movement of water. The observed reduction in the hydrophobic lipid content of the testa layers probably also aids water entry into the seed in a weathered fruit.