Agro-Morphological Characteristics of Weedy Rice ( Oryza Sativa F. Spontanea ): A Precious Genetic Resource for Rice Improvement

Weedy rice ( Oryza sativa f. spontanea ) is considered a feral crop wild relative (CWR) of cultivated rice ( O. sativa ) and has become common weeds of rice fields globally. Weedy rice has been generated either through hybridization or gene flow process between wild rice O. rufipogon and cultivated rice during domestication event. Weedy rice is a conspecific to cultivated rice under the family poaceae which are annual and self-pollinating plant. Weedy rice retains a wide range of diversity in the phenotypic features for adaptation in natural harsh climatic conditions. Many biotic and abiotic stresses tolerance traits have been accumulated slowly through natural evolution to withstand climatic fluctuation. Agromorphological traits were assessed in weedy rice including wild rice and cultivated rice following DUS test protocol for proper characterization and comparative studies. Physicochemical properties such as ASV, GT, GC and sensory based aroma were carried out for six rice genotypes. Phenol test conducted to categories the rice genotypes. Caryopsis ultrastructure was studied using SEM for more clarity in grain fine structural anatomy. Plant height in weedy rice is on average 94.40 cm, and in wild rice it is 120.19 cm. Flag leaf length is 33.69 cm in weedy rice, in case of wild rice it is 21.21cm. Thousand grain weight is 22.50 g in weedy rice whereas in wild rice it is only 13.50 g. Grain per panicle is high in weedy rice (117.10 grain/panicle) but very less in wild rice (39.80 grain/panicle). Weedy rice showed phenol positive reactivity due to presence of PPO (enzyme). Sadanunia was negative in phenol test, is a local aromatic variety. Starch granules mainly CSG ranges from 5.88 to 13.33 μm with irregular spherical structure in weedy rice. In wild rice, CSG is polyhedral structure without any angularity (5.45 μm to 16.26 μm in size).


Introduction
The weedy rice (Oryza sativa f. spontanea) is considered a crop wild relative (CWR) of cultivated rice Oryza sativa, with same genome type AA (2n = 24).Weedy rice is a common weed in paddy fields throughout the world mainly in temperate and subtropical areas. 1 It is commonly known as 'red rice' due to red pericarp colour.Currently, research on the origin and evolution of weedy rice has made some progress, but still there is no definite conclusion about the origin of weedy rice, 2,3 and it is a typical example of convergent evolution in rice crop.It was established that during the time of Neolithic period, approximately it took ancient farmers a few hundred/thousands years to convert wild plants to domesticated cultivars for better yield potentiality and quality food products. 3Very rarely, it was observed that the domesticated crop plants sporadically obtaining again (recapture) some of the habitat similar to wild-like traits at the time of natural evolution. 4This type of evolutionary event is considered as de-domestication phenomenon.This type of phenomenon is also termed as feralization and detected in both the animal and plant kingdoms specifically in livestock species and crop plants, for example found in wheat and rice. 2,5Crop domestication is an important event for transformation of wild species of crop plants into crop varieties through the process of artificial selection by the ancient people (Qiu et al. 2016).Domestication is a unique event through which fitness of a crop species has been enhanced under human cultivation but genetic diversity for the traits has been reduced tremendously in the natural ecological system. 6e-domestication process is a different kind of evolutionary phenomenon where losses of traits are detected (observed) that aggregated during domestication process, as a result domesticated species can be revolved as a self-sustainable 'wildlike' species with low genetic diversity commonly control by natural selection.Adaptability in the natural feral and harsh environmental conditions of the dedomesticated crop varieties remains unclear. 4Newly arisen additional novel mutations may play vital role in environmental adaptation during de-domestication.Wild rice gene(s) may play important role by contributing new mutation/variations which are introgressed into the domesticated crop varieties during the time of domestication and may play important roles for sustainable adaptation to the harsh environmental conditions during dedomestication event through balancing selection.Weedy rice (Oryza sativa f. spontanea) has been converted to an annual crop system commonly pervading the paddy fields are considered as mixed ancestral form of O. rufipogon/O.nivara and cultivated rice O. sativa. 7 was agreeing that weedy rice originated either through the de-domestication event independently from the rice cultivars which was supported by the advance technique of whole-genome sequencing analysis 8,9 or other possibilities of the weedy rice origin may include the process of hybridization system or it can be a gene flow type mechanism occurred between cultivated rice and their wild relatives, whole process can be termed as inter-cultivar hybridization system.Two hypotheses were proposed regarding the origin of weedy rice.One group proposed hat evolution of weedy rice has been occurred through natural hybridization between rice cultivars and wild rice relatives, is called as exo-ferality, 10 other group proposed that weedy rice has been originated from cultivated progenitors in straightway by the process of de-domestication and termed as endoferality. 11Whatever evolutionary phenomena (exoferality or endoferality) are being taking place in this process of weedy rice evolution but recapturing of seed shattering characteristics is the crucial for the origin of Oryza sativa f. spontanea.Genome-wide SNPs variations and domestication-related genes likelihood mechanism support the views that weedy rice is a product of hybridization between cultivated rice and wild rice or it may simply a de-domestication process through which weedy rice has been originated. 9stly, weedy rice embraces intermediate type of morphological characteristics which are descent either from wild rice species (Oryza rufipogon) or cultivated rice (Oryza sativa L.). 12 Following morphological characteristics were demonstrated in the red rice biotypes such as taller height, short growth period, hard and black hull, strong granulation and seed shattering, dormancy period lengthy, long awning, pericarp colour red, variation in panicle size, shorter grain-filling stage and early maturity. 13,14Ultrastructure of caryopsis in red rice has following layers, pericarp, seed coat, nucellus, aleurone layer, and endosperm from outside to inside respectively. 15Colour variation of the rice pericarp due to the accumulation of different pigments such as anthocyanins and proanthocyanidins mainly in the pericarp and seed coat layers.Pigmented rice grains are rich in protein, amino acids, vitamins, vegetable fats, and trace elements such as Ca, Fe, Zn, and Se.Coloured grains also content high amount of biologically active components including flavonoids that have antioxidant, hypoglycaemic properties, other medicinal properties like antitumor activity, free radical-scavenging characteristics 16,17 and protective properties against several noncommunicable diseases, cardiovascular diseases, diabetes mellitus, and metabolic syndrome including cancers.Therefore, consumers are preferring coloured rice for distinctive health benefits. 18Grain colour of weedy rice varies from purple, red, light-red, light green, to white. 19,20edy rice is well adapted in the natural harsh ecological niche with extreme versatile environmental situation because due to the presence of many novel gene(s) which are tolerant to several biotic/ abiotic stresses and may be utilized in rice breeding to improve the released varieties with climate ready potentiality. 12,21Weedy rice genetic resources are considered as novel germplasm for the following characteristics such as tolerance to drought, cold, salinity and showing resistance against many diseases like bacterial blight and blast. 12,14,21,22,23,24It was reported that the resistance activity is genotype dependent and consequently indica type weedy rice shows better resistance in compare to genotype of japonica weedy rice. 22Tremendous phenotypic variation are prevailed in the weedy rice populations and may be the main reasons for high adaptability in the extreme environmental climatic conditions.Huge tillering numbers with extremely expanded root surface makes weedy rice nitrogen use efficient plant and can easily adapted in low resourceful soil. 24Long seed dormancy and seed shattering phenotypic features donate to avoid unfavorable abiotic stresses, leading to escalation the species fitness in the harsh environment.Genetic diversity study in weedy rice revealed that nearly 18% of its genes are associated with tolerance to abiotic stresses including abundant adaptive characters which are not found in the cultivars.As a results they are showing high vigour and reproduction ability in order to make them added tolerant to many biotic/ abiotic stresses. 25us, CWR has been considered a valuable reservoir of genetic diversity for any breeding program.Natural populations of weedy rice are in threatened conditions and leading to diminishing the genepool which needs utmost attention to conserve this virgin genetic resources for future use.Very few germplasm is conserved in the national/international genebank. 7Genetic basis and agro-morphological characteristics of naturally growing weedy rice are not fully studied to understand their eco-habitat.
Therefore, in this present investigation, a comprehensive phenotypic characterization of weedy rice (Oryza sativa f. spontanea) based on agromorphological traits and ultrastructure of caryopsis is investigated, which represent unique source of naturally occurring, highly admixed pre-breeding material for use in rice breeding.This CWR resource is currently available and can be extensively utilized in rice breeding program for varietal improvement which leads to sustainable food security.

Plant Material
Weedy rice at the seedling stage was (Oryza sativa f. spontanea) collected from the adjoining rice field of Mughalsarai (UP) and conserved (on farm) in the experimental rice field of the University of North Bengal for further research work.Wild rice (O.rufipogon) was growing in the ditches of Magurmari river flowing through the University campus near our Experimental rice field and used in this study.Seeds of cultivated rice (Banni, Sadaswarna, Yamuna, and Sadanunia) were collected from farmer's field of Mekhliganj area,Coochbehar district, WB.

Agromophological Characterization Through DUS Protocol
Agromorphological traits were characterized through DUS test protocol (Distinctiveness uniformity and stability test) of PPV&FRA Act (Govt. of India, 2001).

Study the Physicochemical Properties of Rice Grains
Standard method 26 was used to measure the alkali spreading value (ASV) (ranges from 1 to 7 scale basis) to judge grain quality.Result of ASV is low then it signifies the high GT value (gelatinization temperature), equally high ASV value designates a low GT.Aroma was detected using sensory based standard evaluation procedure 27 which varies from 0 to 3 indicator index.Ten good quality seeds of each variety were taken and the husk of each rice seed was peeled with the help of a blade.Bran layer was removed from the grain and were dip in 10 ml solution of 1.7% KOH in a Petri plate.After 10 minutes in the KOH solution, aroma was tested with sensory feeling by us.The aroma is classified into three categories ranging as follows 0, 1, 2, 3 index, non-scented to highly scented respectively.After 23 hours in the KOH solution for alkali spreading test, gelatinization temperature (GT) was measured according to DUS protocol.

Cooking and Retrogradation
Cooking time and retrogradation was determined using common test procedure. 28In this test procedure, unpolished grain was boiled in a test tube and taken out at a definite time interval and pressed in between two glass plates to observe that there is no opaque region.After that the cooked rice was placed on glass plate for retrogradation naturally about 24hrs.

Scanning electron microscopy 2
Caryopsis was cut into round pieces with the help of fine scalpel and one round block was set into the SEM machine for in situ ultrastructure analysis.Histological ultra-structure was detected using a scanning electron microscope (SEM) of Jeol company, Japan (Jeol Model JSM-IT100).Study was conducted at various magnification level with an accelerating voltage of 10 kV. 29,30,31for uncovering the histo-anatomical ultrastructure of the different rice caryopses.Before observing under SEM machine, round piece of caryopsis should be coated with the thin film of gold using a Sputter coater of Jeol Company (Model no.Smart Coater PF 18001006-2).It takes only 2 minutes to finish the gold coating in this Sputter coater because it is used high vacuum evaporator condition to coat the sample.

Phenol Colour Test
Morphological characterization of rice grains is being performed based on phenol colour test (DUS test protocol, Govt. of India).It is being carried out to reveal the reactivity either positive or negative of the grains husk colour. 32,33Therefore, rice varieties can be classified into two main groups either phenol reaction positive (indica type) or negative group (japonica type). 32,33In this test, generally an aqueous (1-2%) phenol solution is used for colour reaction activity with the grain hull.After exposure to this phenol solution for 24 hours, colour variation is determined.In some cases, hull colour changed to dark black colouration but other do not show any change in the hull colour.Phenol positive tested rice varieties are mostly indica type and negative tested rice varieties generally grouped as japonica type.During this 24 hrs experimental time, phenol gets oxidized into dark melanin pigment.Colour conversion occurs due to the presence of polyphenol oxidase (PPO) activity (a positive response) in the seed coat. 34,35Wild Oryza species shows colour reaction if expose to phenol solution and changed to dark brown or black coloration.Indicating that the polyphenol oxidase (PPO) enzyme (tyrosinase protein family) activity is happened. 34,35emonstrating that the Phr1 locus is functioning in the wild rice genotypes and synthesizes PPO enzyme, which ultimately catalyse the dark colour reaction obviously in indica type rice group.Due to loss-of-function mutations at this locus of Phr1, it is not expressing to synthesize PPO in the japonica rice varieties. 35It is showing genetically controlled reaction in the rice varieties.Thus, phenol test is vital test to be used to characterize the rice varieties for identification purposes.

Gel consistency test(GC)
Rice eating quality can be judged by the procedure of gel consistency. 36The 100 mg of milled rice powder were taken in test tubes and wetted by 0.2ml of 95% alcohol containing 0.025 % thymol blue.The 2 ml of 0.2 N KOH was added while shaking in Vortex Genie mixer.The tubes containing samples were transferred into water bath for 8 minutes.After removing the tubes from water bath, it was kept in room temperature for 5 minutes.The test tubes were now kept in ice water for 15 minutes for cooling.Following that the test tubes were placed in horizontally over the graph paper and gel spreading was measured after 30 minutes.Gel consistency are divided into three categories: soft (>60mm in length), medium (40-60 mm in length) and hard (30-40 mm in length).

Results and Discussion
Fifteen quantitative agromorpological traits of six different genotypes (two crop wild relatives and four farmer's varieties) were evaluated based on DUS protocol, Govt. of India (PPV&FR Act 2001).Following traits were considered for evaluation-plant height (PH), flag leaf length (FLL), flag leaf breadth (FLB), panicle length (PnL), grain per panicle (Gr/ Pn), grain length (GL), grain breadth (GB), 1000 grain weight (Gr/Wt), tillering (Till), heading date (HD), maturity time days (MT), yield per plant (YPP), kernel length (KL), kernel breadth and awn (Tables 1, Figs. 1-3).It suggested that there were inherent genetic differences among the six genotypes studied in the present investigation in respect to the morphological traits considered during the analysis.Plant height in weedy rice is on an average 94.40 cm, and in wild rice it is 120.19 cm.Flag leaf length is 33.69 cm in weedy rice, in case of wild rice it is 21.21cm.Thousand grain weight is 22.50 g in weedy rice whereas in wild rice it is only 13.50 g.Grain per panicle is high in weedy rice (117.10 grain/panicle) but very less in wild rice (39.80 grain/panicle) (Table 1).Detail morphological characteristics of thirty-nine traits of six genotypes were summarized (Table 2; Fig. 3) as described in DUS test protocol.Distinctive twenty-three traits of weedy rice were described (Table 3) to know the specific traits for weedyness (Fig. 1).Weedy rice is showing distinguishing morpho-habitat features from that of wild rice (Fig. 1, Tables 1-2).Present characteristics of weedy rice are consistent with the earlier reports (Nadir et al., 2017; Pipatpongpinyo  et al. 2019).Weedy rice panicles are showing variations in awns, hull color, and panicle size, which are supporting the view of previous analysis (Nadir et al., 2017).
Grain morphology and physicochemical properties of the six rice genotypes was studied (Table 4, Fig. 2) for comparative evaluation of the grain characteristics.Highest grain weight (1000 grain) was observed in local rice variety Banni (26.91 g) and lowest in wild rice (13.50 g), whereas weedy rice grain weight was moderate range (22.50 g) (Table 4).Grain length ranged from 8.20 mm, 7.92 mm, 9.21 mm, 7.06 mm, 7.77 mm, and 8.15 mm in weedy rice, wild rice, Banni, Sadaswarna, Yamuna and Sadanunia respectively.Sensory based aroma was detected in wild rice (index-1) and in Sadanunia (index-3), and GC illustrated as soft (ranged from 125 mm to 150 mm), cooked kernel elongation ration was 3.38, 3.60, 3.58, 2.93, 3.99 and 3.50 in weedy rice, wild rice, Banni, Sadaswarna, Yamuna and Sadanunia respectively (Table 4).Spikelet morphology and its different reproductive parts were dissected and characterized (Fig. 4).In the present investigation, it was observed that grain pericarp has diverse colours from red, light red, to white.Similar type of observation also demonstrated by some earlier researchers (Prathepha 2009,  Han et al., 2022).Only Sadanunia showed phenol negative reactivity but remaining five rice genotypes responded to phenol reaction (wild rice, weedy rice, Yamuna, Sadaswarna, Banni) and tested positive (Fig. 5).
Based on phenol colour test, rice varieties may categorize into two groups.Phenol positive indica group and phenol negative japonica group. 33Phenol positive tested rice varieties are mostly indica type and negative tested rice varieties generally grouped as japonica type.During this 24 hrs experimental time, phenol gets oxidized into dark melanin pigment.Colour conversion occurs due to the presence of polyphenol oxidase (PPO) activity (a positive response) in the seed coat. 34,35In this present investigation, five rice genotypes showed phenol positive reaction and categorized as indica type but remaining one genotype (Sadanunia) showing phenol negative test indicating japonica type rice group.Signifying that this variety Sadanunia has a mutation in the locus Phr1 locus, and consequently not able to synthesize functional PPO enzyme.The PPO enzyme activity was not detected in variety Sadanunia.Thus phenol test protocol is an important attribute to identify the varieties for further protection and conservation.Fruit of the rice is caryopsis type where single seed is attached with the wall of the mature ripened ovary (named as pericarp) establishing a seed-like grain.
Rice grain (paddy seed) comprising of two parts, outer protective covering husk (termed as hull) and inner edible part known as caryopsis.
Husk is surrounding by two slices, larger slice lemma and smaller slice palea, which protects the inner edible part caryopsis. 30,31  of polyphenolics compounds are detected in rice bran such as salicylic acid, ferulic acid, and caffeic acid.Among the phytosterols, beta-sitosterol is the most common.All these components have health promoting properties.Different amino acids, many cofactors and various type of secondary metabolites available in the rice bran with medicinal values and considered as a whole food. 17dosperm layer mainly composed of starch, starch granules may be polygonal or spherical in shape and size.-10).Specifically, the shattering seed habit and long dormancy period acquired by the feral weedy rice is a unique observation in this present investigation which is consistent with the earlier report. 37,38Pericarp colour is ranging from red, greenish, brown, that report also consistent with previous study. 39

Conclusion
There was no report about the agro-morphological features of weedy rice (O.sativa f. spontanea) based on caryopsis ultrastructure using scanning electron microscopy (SEM).In the present study, comparison was made with respect to wild rice (O.rufipogon) and cultivated rice varieties (Yamuna, Banni, Sadaswarna, Sadanunia) for characterization of the rice germplasm.Germplasm characterization is one of the most important parameters for any breeding program to utilize as parental material.Agromorphological traits and physicochemical descriptions were considered to characterize the six rice genotypes including this weedy rice.Weedy rice (Oryza sativa f. spontanea), is an annual, self-pollinating plant and considered as feral conspecific to cultivated rice (O.sativa).Weedy rice appears to possess a wide range of variation in the phenotypic characteristics for adaptation in natural harsh climatic conditions.Many biotic and abiotic stresses tolerance traits have been accumulated slowly through natural evolution to withstand in the climatic fluctuation.Therefore, it needs conservation through on farm in situ process and utilization in the breeding program to develop climate resilient high yielding improved rice varieties with quality grain for sustainable food security.

Fig. 1 :
Fig. 1: Morphological characteristics of weedy rice, and wild rice were represented.A: Wild rice Oryza rufipogon growing in pot, B: Inflorescence of O. rufipogon with anthers and stigma, C: Panicle shape and size of O. rufipogon.D: Grain morphology of O. rufipogon.E: Weedy rice (O.sativa f. spontanea), F: Panicle shape and size of O. sativa f. spontanea, G: Grain morphology of O. sativa f. spontanea and H: Natural habitat of weedy rice (O.sativa f. spontanea).

Table 5 : Summary of the caryopses ultrastructural characteristics studied under scanning electron microscope (SEM) of the six genotypes (CWR and farmer's varieties) was depicted.
Thin cell wall contains very little amount of cell wall materials of the endosperm layer.Shape of the CSG was polyhedral to spherical with different size (3.53 μm to 23.07 μm) with minimum angularity and surrounded by many numbers of small size protein bodies (PB) (0.692 to 1.53μm).Compound starch granule (CSG) ranges from 5.88 to 13.33 μm with irregular spherical structure (Table 5: Figs.7-10) in weedy rice.In wild rice, CSG is polyhedral structure without any angularity (5.45 μm to 16.26 μm in size).Microspore-pinhole was also present in all six rice genotypes (Figs.7-10).CSG are various shape and size, spherical to polyhedral with moderate angularity (3.53 to 13.748 μm in size) in Banni.PB is moderately present with less impression of PB (0.692 to 1.53 μm in diameter) in all the rice genotypes (Table 5).It was detected that CSG ranges from polyhedral to spherical in shape and size from 3.53 to 23.07 μm (Table 5; Figs.7-10).Both wild rice (O.rufipogon) and weedy rice (O.sativa f. spontanea) have long awn with barbed features (329.169 to 358.489 μm) (Fig. 6).Bran thickness was highest in Banni (37.029 μm), followed by Yamuna (36.327 μm), wild rice (30.85 μm), weedy rice (29.55 μm), Sadaswarna (27.682), and Sadanunia (25.03 μm) (Table 5; Figs.7