Florogenesis and Female Gametophyte Development in Allium cepa L. cv. Krishnapuram

Florogenesis is one of the most complicated and interesting processes in the nature. This process involves developmental, physiological and molecular events leading to transformation from vegetative to reproductive phase for optimal seed production and the continuation of species. The basic knowledge about flowering processes, male and female sexual systems support basic and applied research and breeding programs. Most of the onion varieties from India are short day varieties, more diverse than other exotic germplasm and useful as a source of new alleles for supporting breeding programs. The present investigation was focused to study for the first time florogenesis process by scanning electron study for the first time florogenesis process by scanning electron microscopy (SEM) and development of female gametophytes by light microscopy in order to acquire basic knowledge useful for optimizing in vitro process to produce gynogenic haploid to support and speed breeding program in short-day onion Allium cepa L. cv. Krishnapuram (KP) or Bangalore Rose. This study revealed that shoot primordium differentiated into inflorescence meristem in the month of December, while seeds were planted in the field in the September. The individual florets are preceded by a variying number of floral initials. The female gametophyte developed from chalazal side megaspore. The embryo sac development is a bisporic Allium type showing shortlived antipodals. The histological study suggests that the use of big or preanthesis flower buds with embryo sac for production of gynogenic haploids to support breeding program in onion cv. Krishnapuram (KP). However further studies are needed for confirmation of this observation.


Introduction
The genus Allium (family: Liliaceae) comprises about 500 species distributed in the Northern hemisphere. The Cepa (Mill) Prokh section of Allium consists of nine wild species such as A. cepa (commonly known as bulb onion) and A. fistulosum (bunching onion). Onion is a historic vegetable crop considered to be originated in Central Asia and has been grown all over the world. It is cooked to combine flavor and served fresh in salads in the preparations of gastronomy.
Traditional breeding and genetic methods have been employed to enhance onion quality, yield and tolerance to environmental stresses. However onion breeding efforts are hampered due to its biennial life cycle, heterozygosity and higher inbreeding depression. There is an urgent need to speed and support onion breeding programs to meet the consumer demand in the context of population pressure and climatic change [1]. To face with these problems, onion breeding programs should be integrated with recent plant molecular, biotechnology and omic techniques [2].
Florogenesis is one of the most complicated and interesting process in the nature. This process involves various developmental, physiological, biochemical and molecular events leading to conversion from vegetative to reproductive phase for optimal seed production for the continuation of the species. The basic knowledge about flowering process and development of sexual systems support basic and applied research and speed conventional breeding programs [2]. The florogenesis process is conveniently classified into five successive steps: induction, initiation, differentiation (organogenesis), maturation and development of floral parts and anthesis [3]. Flowering of diverse taxa within the genus Allium is greatly varied as per their developmental biology, morphology, genetic control and interaction with the growing environment [4]. The different biomorphological groups respond variedly to inductive conditions and thus flower in different ways. The process of florogenesis has been studied in very few species of Allium such as ornamental alliums [5], shallot [6] and garlic [7].
Both flowering and bulbing processes are important traits in A. cepa influenced by vernalization and photoperiodic responses, respectively [8]. With regard to temperature requirement, onions require cool temperatures during juvenile phase and warmer temperatures during maturity. Onion varieties are generally classified as short-day and long day varieties on the basis of the photoperiod.
Various reports have indicated that long day and short-day germplasm are genetically dissimilar. These two groups further subdivided on the basis of geographical origins [9]. Moreover, it is found that most of the onion varieties from India are short-day varieties, further varied than other exotic germplasm and useful as a source of new alleles for breeding purposes [10]. The differences in the length of the juvenile phase (physiological age), the response to photoperiod and temperature required for floral induction have been already reported with long-day and temperate onion varieties [11].
Gynogenic haploids have been extensively and routinely used to support and in vitro culture using explants such as non-pollinated ovules, ovaries or flower buds [12]. It was also found that the developmental stage of explants is one of the critical aspects influencing gynogenic haploid production. The female gametophyte at different developmental stages has been used for induction of gynogenic haploids. Of the various stages tested. The mature embryo sac stage is proved to be more suitable than other developmental stages [13]. The analysis of the structure and development of the onion inflorescence and individual flower has been already reported [14]. Only one study could be found that was centered on the florogenesis process in long-day variety of onion [15]. However, these authors neither describe nor report the florogenesis and female gametophyte development in short-day varieties of onion. The present investigation was focused to study florogenesis process by scanning electron microscopy (SEM) and female gametophyte development by light microscopy in order to get basic knowledge useful for optimizing in vitro protocols to produce gynogenic haploids for supporting breeding programs in short-day onion cv. Krishnapuram (KP) or Bangalore Rose.

Scanning Electron Microscopy
For scanning electron (SEM) examinations, umbels were fixed in 2.5% glutaraldehyde and 2% para formaldehyde in 0.

Microtomy and Light Microscopy
To study megasporogenesis and megagametogenesis ovaries from flower buds at four different stages of development [small, medium, big and pre-anthesis] were analyzed. Flowers were fixed in Carnoy's fluid (ethanol: acetic acid, 3:1) for 24 h at room temperature and then stored in 70% ethanol. Longitudinal serial (LS) sections were prepared according to standard techniques of wax embedding [16]. Embedded ovules were sectioned by rotary microtome for semi-thin sections. The staining of sections was carried out with safranin and toluidine-blue-O. The microscopic studies were examined under Carl-Zeiss (Germany) microscope. Micrographs were taken with CCD camera (Prog Res C3 Jenoptik).

Results and Discussion
The cotyledon, which pushes the embryonic rootlet downwards. One to two days afterward, the upper part of the cotyledon, grows an inverted U shape loop which is move forwarded upwards through the soil surface by elongation of two sides of cotyledon. In generally, onion crop is propagated by seeds, bulbs or sets.
When grown from seed, the plants reported to go through vegetative and flowering stages of similar general appearance. The principle stages related to the growth of onion and other edible alliums from seed already reported by [17].
The earliest stages of development from seed are distinguished using the term loop, crook and first and second leaf stages. Where as in bulb propagated crops like garlic, shallot and rakkyo, the first leaf to report from the bulb penetrate to the soil surface is bladeless [18]. During vegetative growth, the stem is flattened to form a disc at the base of plant. At the top center of stem disc in the shoot apex, from which leaves are initiated oppositely and alternately (Figure 1(b)). Each leaf consists of blade and sheath. The sheath develops to completely encircle the growing apex. At the junction of the blade and sheath there is an opening or pore when the tip of the blade of the next younger most leaf can be seen. The blade of younger leaf was elongated and emerging through this pore. The apical meristem of vegetative shoot is flat and leaf primordial develops from the periphery towards centre. As new leaves are initiated and expand near the shoot apex, older sheath bases get pushed further away from the apex by continuing lateral expansion of the disclike stem. The vegetative growth of stem remains till the end of the November. Thereafter, plant enters into the reproductive stage at 10 -14 leaves stage. The onion Krishnapuram variety remained in vegetative stage for a period of 2 -3 months i.e. from mid September to end of the November.
When grown from seed, all Allium plants require to reach a certain physiological age (for critical mass) prior being capable of florogenesis and blooming. The length of the juvenile phase is varied according to the species, it ranges from a few months in bulb onion [11], chives [19], Japanese bunching onion [20], leek [21] and shallot [11] to 5 -6 years in A. giganteum and A. karataviense [22]. The duration of juvenile phase depends on the genotype as well as the growth environment. Both factors control the amount of stored reserves necessary for successful blooming. In addition to the available reserves, the ability to form flower also depends on the size of the apical meristem [3]. Further the transition from vegetative to the reproductive stage normally happens in the first or second growing season after the formation of 10 -14 leaves in seed propagated alliums [11]. Under inductive conditions, floral initiation in shallot [6] and leek [21] is already possible after development of the first 6 and 7 true leaves (including leaf primordia), respectively. The shoot apical meristem swells to form a dome shaped reproductive meristem in the month of December (Figure 1(c) and Figure 1(d)). SEM photograph   (Figure 1(e) and Figure 1(f)). After blooming, plant starts senescence and also complete bulb formation in the field ( Figure 1(g)). The developmental process of umbel studied in seven stages as shown in Figure 1(h) by Scanning Electron Microscopy (SEM) based on the size, differentiation and development of flower buds (Figure 2(a)). The reproductive meristem was enveloped by the leaf like and papery spathe. The spathe consists of several membranous bracts (Figure 2(b)), which arises as close to uniform ring and elongates quickly to form more than 30 cm long inflorescence stalk known as scape. The scape is commonly hollow when mature, round in cross section and somewhat swollen at the middle or near the base. Similar observations were already reported in Allium sp. [1]. The proper elongation of scape elongation is essential for a successful bloom. This process strongly influenced by the storage and growth temperatures, as well as the photoperiod [23].
In garlic when the floral stalk attains 15 cm in length, the pedicles elongate and the inflorescence becomes spherical [11].  (Figure 2(h)).
This flower opening was observed from oldest to youngest flowers within each cyme and over the whole surface of the umbel as reported earlier by [11]. The flower colour and the pattern of opening of individual flowers within the umbel vary with species. In shallot floral morphology is very similar to that of bulb onion, a clear direction of primordia differentiation in individual flowers has not been observed [6]. In garlic when compared with in onion and shallot, each perianth lobe and the subtended stamen arise at the same time from a single primordium [7].

Female Gametophyte
In A. cepa Krishnapuram onion has a trilocular ovary, and syncarpous gynoecium, which have superior ovary with two ovules on axile placentattion. Style is short and filiform and the stigma is minute. Based on the four different developmental stages of flower buds, the process of megasporogenesis and megagametogenesis was studied by using ovules (Table 1).

Megasporogenesis
The ovule consists of three basic structures such as a megasporangium, two integuments (inner and outer), and a funiculus, that are connected to the ovary wall. Early in the development of the pistil, ovular primordium and ovary wall differentiate simultaneously. The ovular primordium is composed of a group of meristematic cells located in the center (Figure 3(a)). Soon as archesporial cell, a large cell with dense cytoplasm is differentiated immediately below the epidermis. The periclinal division of archesporial cell leads to the formation of primary parietal cell and primary sporogenous cell. There after a megasporocyte or megaspore mother cell (MMC) formed from the primary sporogenous cell. The MMC can be distinguished among the other nucellus cells at a very early stage , even before the integuments begin to form at its base. The parietal cell divides periclinally 1 -2 times to form 1 -3 layers of nucellarcells, the ovule therefore is crassinucellate (Figure 3(b)). After differentiation, the inner integument is positioned at the nucellus, while the outer integument is located above the inner in-        (Figure 3(c)). Cell wall formation after meiosis I of megasporogenesis seems to be of universal occurrence in all plants [26]. The functional megaspore mother cells at the stage of development fully surrounded by callose ( Figure 3(d)). In case of A. consanguineum a species growing wild in the Western Himalayas, the cell wall is not laid after the first meiosis [27]. The degeneration of the micropylar daughter cell in A. cepa is characteristic of many species of Allium and occurs in other genera as well. The origin of the embryo sac from the chalazal daughter cell has also been described for A. fistulosum [28], A. porrum, A. victoriale, A. paniculatum, A. flavum, A. ursinum, A. edbdaneese, A. uniflorum, A. rotundum, and A. sphaerocephalum, A. carnatum, A. oleraceum, A. scorodoprasum, A. moly bulbiferum, A. paradoxum, and A. sativum [29].

Megagametogenesis
Megagametogenesis process begins with increase in the size of the female gametophyte. The mitosis-I division of two megaspores nuclei produces four nuclei, two situated at the chalazal end and the other two at the micropylar pole. These nuclei were alienated by a large vacuole in the centre of the embryo sac without cytokinesis (Figure 3(e)). Later on these four nuclei go through mitosis-II to gives rise eight nuclei. The four nuclei are positioned at the chalazal end and the four at the micropylar end. The mitotic division I & II occur in a highly coordinate manner at both ends of the embryo sac (Figure 3(f)). Two successive mitotic divisions and absence of cytokinesis in long day varieties of onion. In general, the cellularizedbisporic embryo sac contains seven cells (eight nuclei): three antipodal cells situated at the chalazal end, two polar nuclei and three celled egg apparatus [14]. However, we did not observe the presence of antipodals in the embryo sac. Further observations revealed that the egg apparatus is lied in close contact with the micropyle (Figure 3(g) and Figure 3(h)). Several variations were reported in Allium type of embryo sac development, [30]. The absence of the antipodals may be due to their rapid degeneration or may be unnoticed as they concealed at the end of chalazal side [26]. In other varieties of A. cepa, the presence of antipodals has not been reported in all published papers [12]. The existence of short lived antipodal cells has also been reported in Chichoriumintybus [31] and Agave tequilana [32]. In addition to that, the close contact of the egg apparatus with the micropyle is highly similar to what was observed in the ovular apparatus of Ornithogalum caudatum [33]. Moreover, as observed in many other angiosperms, the egg cell possesses a highly dense nucleus placed towards the chalazal extreme of the embryo sac [34].

Conclusion
Seeds of A. cepa L. cv. Krishnapuram onion used for bulb production in the field in two seasons Rabi and Kharif during, September-April and May to November, respectively. It was found that the primordium of the inflorescence axis differentiated in the month of December while seeds were planted in the field in September. The individual florets are preceded by a varying number of floral initials in the month of December. The female gametophyte was developed from chalazal side megaspore. The embryo sac development is a bisporic Allium type, showing short-lived antipodals. The observations reported here show basic information on florogenesis process and the female gametophyte development to support breeding programs. The gynogenic response is strongly depended on the developmental stage of female gametophyte. The present histological study correlated with the developmental stages according to the size of the flower buds (small, medium, big, pre-anthesis) ( Table 1). Gynogenic development start only after a mature embryo sac has been organized. This observation might suggest the use of big or pre-anthesis flower buds with embryo sac for the production of gynogenic haploids to support breeding program in onion cv. Krishnapuram. However, further studies are needed for confirm this observation.