Comparative ultrastructure of caryopsis and leaf surface anatomy in wild rice Oryza coarctata and O. rufipogon through Scanning Electron Microscope (SEM)

The wild rice Oryza coarctata (Roxb.) is an herbaceous halophytic plant belongs to the grass family poaceae prevalent to the coastal regions of Southern Asia. The O. coarctata is the only hydro-halophytic rice germplasm under the genus Oryza and shows high salinity. Caryopsis ultrastructure of O. coarctata was compared with another wild rice O. rufi pogon through Scanning Electron Microscopy (SEM) including leaf surface anatomy to unveil the differentiation between the two species of Oryza. In O. rufi pogon, embryo is small and orthodox type with long viability, in O. coarcata, embryo is large size and recalcitrant type. In O. coarctata, lower part of the spikelet has callus and expanded disc-like structure, without any globose rachilla, lemma devoid of tubercles, prickles and microhairs. Leaves of O. coarctata contain salt hairs and salt glands to secrete excessive salt, during high salt concentration which is a most important characteristic of this halophytic wild rice. Caryopsis endosperm contains starch granule of spherical shape with protein bodies in O. coarctata, whereas polygonal or hexagonal with moderate angularity starch granule in O. rufi pogon. Aleurone layer is not so distinct in O. coarctata in compared to O. rufi pogon, where it is clear and distinct. Protein profi ling was studied through SDSPAGE for banding pattern variation analysis. This study of rice caryopsis ultrastructure and leaf surface anatomy including salt-hairs will contribute to the knowledge about the conservation of such precious germplasm of Sudarban mangrove region for the improvement of climate resilient rice varieties in future through pre-breeding and transgenic system. Research Article Comparative ultrastructure of caryopsis and leaf surface anatomy in wild rice Oryza coarctata and O. rufi pogon through Scanning Electron Microscope (SEM) Subhas Chandra Roy1* and Anurag Chowdhury2 1Plant Genetics & Molecular Breeding Laboratory, Department of Botany, University of North Bengal, PO-NBU, Siliguri-734013, WB, India 2Sundarban Biosphere Reserve, 24 Pargana South Division, Pin-700027, WB, India Received: 25 March, 2021 Accepted: 08 April, 2021 Published: 12 April, 2021 *Corresponding author: Subhas Chandra Roy, Plant Genetics & Molecular Breeding Laboratory, Department of Botany, University of North Bengal, PO-NBU, Siliguri-734013, WB, India, E-mail:


Introduction
The tribe oryzeae consisting of 12 genera, among these only 2 are cultivated species and remaining 22 are wild species. Total 24 species are available under this tribe Oryzeae and spread worldwide [1,2]. Among the two cultivated species, one species Oryza sativa L. grows throughout the world and O. glaberrima Steud. only grows in West Africa [3,4]

Species of this genus
Oryza are distributed through the world (Asia, Africa, Australia, and the Americas) ranges from driest desert to sea level to an altitude of 3000 m above sea level. These species are growing in such diverse agro-climatic conditions and show wide range of genetic diversity because they have not undergone any human rice varieties for yield increase and better performance in the different ecoagroclimatic conditions. It is uncertain whether the current agricultural production system will be able to feed the expected nine billion people in this world by 2050 [7][8][9][10], whereas we need to produce double amount of rice. Because, productivity of rice affected several biotic and abiotic stresses (drought, cold, fl ood, salinity, heat) due to climate changes [11,12]. Plant has evolved some kind of mechanism to protect from such type of abiotic stresses by a series of adaptation leading to morphological, physiological and biochemical changes [13]. Wild relatives of the cultivated species are the natural source of 'tolerance genes' against these stresses (Abiotic) [14]. Salinity stress may cause 20% yield decrease in rice production [15] and may cause many physiological changes ( Na+/K+ ratio balance) in plant growth and development [16][17][18][19]. Abiotic stresses such as fl ooding, drought, salty soil and other climate change has created more burdens to food security [20].
The Oryza sativa gene, OsHKT1;5 is the causative genetic component of The Saltol is a major major Quantitative Trait Locus (QTL) containing gene OsHKT1;5 and is necessary genetic component for salt tolerance in rice [21]. It has identifi ed as a plasma membrane transporter which controls the partitioning of Na+ between roots and shoots through effl ux of Na+ from the xylem to neighbouring cells of parenchyma [22]. Local rice cultivar indica type, Nona Bokra has high salt tolerant potentiality due to increased Na+ effl ux activity in presence of OsHKT1;5 transporter gene with four amino acid changes in compared to other salt sensitive rice cultivar [22,23].
Improved rice varieties can be developed by exploring and proper utilizing the rice germplasm for desirable abiotic stress tolerant traits [24]. Salinity of the soil is increasing globally day by day due to surface irrigation, high cropping intensity with high yielding varieties resulted in reduced yield potentiality [25]. Genetic potential of stress tolerance (salt) trait is existing among the germplasm of primary gene pool are utilized for the development of salt tolerance crops. Wild species of rice is a good source of many biotic and abiotic tolerance genes /QTLs which are not selected during rice domestication [26]. It has been reported earlier that Oryza coarctata, a wild rice species of mangrove ecology has high adaptability to saline stress [27], whereas other wild species of rice such as O. ridleyi and O. schlechteri are known for the trait of submergence tolerance [6,17,28]. The O. coarctata is considered as hydrohalophytic because they can tolerate high soil salinity and naturally growing in mangrove region of the world carrying allotetraploid genome set KKLL (2n=4X=48) with self-fertility behaviour and also has submergence tolerance trait [4,[29][30][31] [17,32]. This wild rice can be a good source of germplasm to develop high salinity tolerance improved varieties [32]. Hybridization was made between O. sativa and O. coarctata to develop hybrid F1 to establish their cross compatibility with cultivated rice [33] and others (www.irri.org). Salt tolerant rice cultivars with introgressed O. coarctata traits for salt tolerance are currently under development in the International Rice Research Institute (IRRI; www.irri.org). Normal growth was unaffected at high salt concentration (400 mM of NaCl) but growth of cultivated rice (O. sativa) was hampered [34]. The wild rice O. coarctata has unique leaf morpho-anatomy and physio-molecular mechanism by which they exclude salt (Na+) from the leaf surface using salt hairs and salt glands [17,[34][35][36]. It is signify that O. coarctata keep away from Na + toxicity in mesophyll cells by compartmentalization of salt in the vacuole and in epidermal hairs maintaining a low Na:K ratio, a mechanism generally found in halophyte grasses [17,34]

Scanning Electron Microscopy (SEM) for the Caryopsis ultrastructure analysis
Mature caryopsis was taken out by removing the husk using fi ne forceps. Caryopsis cut in to round pieces of 1mm thick length wise by applying slight pressure on the middle of the caryopsis with a razor blade. Fractured surface (solid round ring) facing upwards to get whole inner surface (in situ condition) of the grain was mounted on a specimen stub and coated with thin fi lm of gold by means of a sputter coater (Jeol Model Smart Coater PF 18001006-2) about 2 minutes at high vacuum evaporator condition. Detailed inner structure was viewed through scanning electron microscope (SEM) (Jeol Model JSM-IT100, Japan) at various magnifi cation with an accelerating voltage of 10 kV [41,42] to study the histoanatomical ultrastructure of the rice caryopsis of O. coarctata and O. rufi pogon. Mineral elements are to be qualitatively detected using EDAX system of SEM mainly zinc, and iron.
These elemental defi ciencies may cause hidden hunger.

Protein profi ling through SDS-PAGE Electrophoresis
Protein profi ling was carried out using SDS-PAGE electrophoresis based on standard protocol [43]. Leaf sample (1 g) was taken for protein extraction and purifi cation. Phosphate buffer pH 7.5 was used for protein isolation from the tender leaf of wild rice O. coarctata and O. rufi pogon. SDS-PAGE analysis was performed using isolated protein from wild rice. The analysis was performed using a SDS-Tris-glycine buffer system with 5% (w/w) stacking gels and 12% (w/w) resolving gels using the standard method [43]. Electrophoresis was initially performed

Plant morphology and leaf surface anatomy
The wild rice Oryza coarctata (Roxb.) is naturally growing as associated vegetation in the mangrove forest of Sudarban Biosphere Reserve area of the district South 24-Parganas, West Bengal, India, which is salt tolerant hydro-halophytic species belongs to the family poaceae under the genus Oryza ( Figure 1). This wild rice O. coarctata is morphologically distinct from other species of wild rice Oryza rufi pogon ( Figure 1). salt hairs are arranged all over the leaf surface in ridges and furrows, fi nger like salt hairs only distributed in the margins of furrows ( Figure 3D,E). Leaves are lanceolate in shape, not leathery; margins are not tuberculate, main vascular bundle is prominent in case of O. rufi pogon ( Figure 1). Both type of salt exclusion structures (salt glands and salt hairs) are found in O. rufi pogon too ( Figure 4).
The infl orescence is spikelets type and mature fl owers are present on the upper end of the spikelet (non-fl attened) in O. coarctata. Panicle is up to 20 cm long. Fertile lemma and palea are smooth in surface with any special types of features. The spikelets are narrowly oblong to narrowly ovate, 12.5 to 14.5mm long (including the awn), and obliquely articulated with the pedicel (Figure 2). A unique callus is expanded as a rigid disk-like appearance which is transversely narrowed. Glumes are linear subequal, one-third as long as the spikelet, lacking apparent epidermal characteristics. The lemma apex bears a rigid small awn of about 4 mm long, with microhairs and stomata. Apicular knobs are not present on the lemma apex.
Globose rachilla is lacking in this O. coarctata. The lemma is 7-9 nerved and the palea fi venerved (O. coarctata) but others species have fi ve-nerved lemma and three-nerved palea. In case of O. rufi pogon, spikelets are thin linear ranges from 7.95 mm to 8.52 mm long, somewhat smaller than the O. coarctata. Details comparative description about the plant morphology, leaf morpho-anatomy, caryopsis morpho-ultrastructure between two wild rice O. coarctata and O. rufi pogon has been summarized in a tabular outline (Table 1)

Caryopsis ultrastructure under SEM study
The whole kernel, caryopsis of O. coarctata has an unusual large embryo (4.5mm to 5mm) relative to the endosperm (8.8mm to 9.5mm) (half the size of the caryopsis) with a somewhat bent apex, and a short petiole-like attachment at the base ( Figure 5). Embryo is narrowly elliptic and 4.5 to 5.0mm long. This large embryo has free epiblast large in size and found a distinct cleft like structure in between the coleorhiza and the scutellum (Table 1; Figure 5) but lacks auricles. In contrast to this embryo of O. coarctata, small size embryo (1.0 to 1.5mm long) is observed in O. rufi pogon (one-fi fth to one-third size in this wild species) without any cleft or free epiblast (Table 1,  Leaf has ridges and furrow, upper surface has many salt gland and salt hairs, salt gland are present throughout the upper surface, salt hairs only on the marginal side of furrows. Salt gland varies 2.8μm to 6.4μm in diameter, salt hairs ranges from 5.72μm to 18.56 μm in length. Stomata range from 28.718μm to 30.4μm in length and 11.40μm to 12.241μm in width only on lower surface. Lanceolate, not leathery, margins not tuberculate, main vascular bundle prominent. Leaf surface with different cellular structure, parallel arrangement of dumbbell shaped cellular features and parallel arrangement of salt gland and salt hairs (fi nger like projection) on the upper surface. Size of the dumbbell cell ranges 18.88 μm in length and 9.141μm in width, in between this structure, a small salt gland like structure was observed (3.601 μm in diameter). This dumbbell feature is totally absent in O. coarctata. Salt gland ranges 3.52 to 4.46μm in diameter, salt hair is 5.625 to 6.47μm in long.

Spikelet
Spikelets are narrowly oblong or ovate, length varies from 12.5 mm to 14.5mm, width 3.5mm 4.2mm, lower part of the spikelet with callus expanded and disc-like, rachilla lacking, glumes present, lemma and palea normal in size, small microhairs, stomata, and papillae in lemma, small awn 4 mm to 5 mm is present at lemma tip; awn base has stomata, some specifi c structure such as tubercles, microhairs, and prickles on lemma-palea are lacking in this species.
Husk colour is yellow.
Spikelets are linear thin, length ranges from 7.95mm to 8.52mm and width 1.93-2.11 mm, lower part of spikelet showing callus with lateral articulation scar, glumes (offset) with silica bodies, globose rachilla present, lemma, and palea are normal, lemma with tubercles, microhairs, and prickles, long awn ranges 30 mm to 90 mm with 0.25 to 0.30 mm in diameter.
Husk colour is blackish.

Caryopsis
Caryopsis is large, length varies from 8.8 mm-9.5 mm, width 1.5 mm to 1.80mm with petiole like structure at base (0.5-0.6mm long) and tuber like bent apex; diameter of the caryopsis varies 1.526μm to 1.588μm. Caryopsis upper surface is rough uneven of irregular rectangular series of small groove (27.665μm to 1.880μm in size) with protein bodies (4.419 μm diameter), which is unique trait of O. coarctata. Endosperm parenchyma cells are irregular in shape and size ranging from ovoid to rectangular (98.05μm x 67.930μm), starch granule is spherical in shape, SSG varies from 9.136μm to 11.991 μm in diameter, CSG is 11.991 μm to 17.140μm in size, PB (0.576μm to 1.04μm in diameter), cell wall is thick 0.7μm to 0.9 μm in thickness. Caryopsis colour is black. Length 6.10mm to 6.5mm, width 1.75mm to 1.95mm with no petiole like structure. Upper surface is reticulate type with irregular pattern rectangular markings (20μm x 12μm in size). No protein bodies on the surface of caryopsis.
Endosperm parenchyma cells are not very prominent, ovoid to rectangular in shape with very thin cell wall. Starch granules are polygonal to hexagonal with medium angularity (CSG is 5.64μm to 16.50 μm in size, SSG is 3.214μm to 5.712μm in size) associated with protein bodies (1.40μm -4.30μm in size).

Embryo
Large embryo relative to endosperm (4.5mm to 5.0mm)almost half the size of the caryopsis, cleft between coleoptiles and coleorhizae.
Small embryo relative to endosperm (1.0-1.5mm) one-fi fth to one-third size.
No cleft in the embryo. Dutt 1986) through salt gland and salt hairs. This halophytic wild rice O.coarctata is a saltloving rice species which is able to remove the excess salt through salt glands by the coordination action of various genes [45]. It is facultative halophytes but requires saline environment for initial growth establishment. Therefore, it can be utilized as an important source of genes/   alleles and QTLs for salinity and submergence tolerance [46].
In general, O.coarctata is salt tolerant wild rice genotype of saline coastal region of West Bengal and is regarded as a gold mine of abiotic stress tolerant germplasm specifi cally to study the salt tolerance mechanism and identifi cation of genes which can be used for rice improvement against salinity stress.
This halophytic wild rice belongs to secondary gene pool of rice  Figure 2). The present fi nding is consisted with the observation of earlier study [48]. Embryo is large in size Single Starch Granule (SSG) ranges from 9.14 μm to 11.99μm in diameter, these are loosely arranged (piled up one after another)

with void spaces within the parenchyma cells and Compound
Starch Granule (CSG) varies from 11.99μm to 17.14μm in diameter ( Figure 5)  also imperative to emphasize that isolated populations within species may also hold crucial genes [2,6]. Geenetic diversity within the wild rice species is an essential resource to improve rice production under climate change scenario mainly abiotic stress tolerance [12,50]. Transfer of desired traits from the wild species to cultivated species is not so easy because these wild species are connected with several unwanted weedy traits, like grain shattering trait, poor plant type, poor grain characteristics and very low seed yield. In addition to that, several incompatibility barriers limit the transfer of desired traits related genes from wild species into cultivated species.

Conclusion
The Oryza coarctata (Roxb.) is an extremely halophytic wild rice species highly tolerant to extreme salinity and can withstand salinity levels as high as sea water up to 300-400mM NaCl is perennial herb up to 2 meter tall with branched stalk, extremely hydro-halophytic with extensive rhizome system. Leaves are waxy leathery (coriaceous) that are strongly ribbed, with special type of salt glands and salt hairs for the exclusion of excessive salt from the cellular environment to thrive from salinity stress. Embryo size is large with free epiblast including cleft in between the coleorhiza and the scutellum. Embryo is short lived and recalcitrant in nature.
It has an unusual large embryo with a large free epiblast and a distinct cleft between the coleorhiza and the scutellum, short lived recalcitrant in nature. Caryopsis has a bent at tip and petiole like structure at the base. Lemma and palea of O. coarctata is lacking any kind of special features on upper surface mainly tubercles. It is found in all other species of Oryza, a distinctive epidermal out growth and considered as a synapomorphy which support the view of monophyly of the genus Porteresia previously.
Wild species of rice serve as an untapped reservoir of genetic diversity containing agronomically valuable traits that can be used to develop new rice varieties through breeding program. The mangrove associated wild rice O. coarctata can be a good source of salt tolerance gene(s) or QTLs to provide salinity tolerance to improved rice varieties through conventional breeding or through transgenic technology. This salt tolerance species can also furnish C4 like photosynthetic apparatus and mechanism to the C4 rice consortium to develop C4 rice plant to increase the yield potentiality to meet up the food grain demands in 2050 for more than >9 billion people. Breeders and genetic engineers should look in this species very carefully to utilise its C4-like anatomical features and molecular mechanism of photosynthesis in the salinity environment (abiotic stress conditions). Its recalcitrant type seed longevity might be due to irregularities found in the Bran layer components. The bran layer commonly comprises of three distinct layers in other species of rice-pericarp, testa and aleurone layer (altogether called as bran layer). In this species of O. coarctata, aleurone layer is not prominent and does not contain any kind of aleurone grains (AG) like other species. Aleorone layer of the rice bran layer play vital role in seed germination and seed viability. Due to nonperforming aleurone components in the species O. coarctata seed is recalcitrant short lived. Ultrastructure of the caryopsis of O. coarctata (reported fi rst time) has been described in details to provide genetic diversity and variation of the inner histo-anatomical features of such unique traits, which is distinct from that of the other rice species O. rufi pogon. Bioprospecting of such genes and proteins coupled with genomic and proteomic approaches remain an exciting area of research in evaluating this plant as a model for salt tolerance for the rice plant.

Authors contribution
SCR was a major contributor in writing the manuscript, proposed the research idea, conceptualized the research design and prepared the fi nal manuscript including the original experimental work, and data recording. AC was involved with material collection, location survey and habitat study, at Sudarban Biosphere Reserve, WB, India.