FIELD OF INVENTION
[001] The present disclosure broadly relates to the field of plant biotechnology. The present disclosure discloses recombinant construct, recombinant vector, recombinant host cell, and methods for improving grain quality.
BACKGROUND OF INVENTION
[002] Rice is one of the most important staple food crops for more than half of the population in the world. The quality of rice is one of the key determinants of its saleability in the market. Thus, breeders are mandated to develop germplasm of high quality that suits the particular needs of the market being targeted. There are several defined classes of rice, based on the physical appearance of the milled rice, the cooking properties, and the aroma of the rice. The traits that are considered important in determining the physical quality of grain are length, width and uniformity and weight, head rice yield, colour (whiteness and translucence), chalk, and cracks. The length and width of a rice grain are important attributes that determine the class of rice. There are three main classes of rice, based on grain length: short, medium, and long. The ratio of the length and the width is used to describe the shape and class of the variety. The other important aspect of length and width is uniformity. Further, the grain weight provides information about the size and density of the grain. Uniform grain weight is important for consistent grain quality. Head rice yield is the weight of whole white rice grains remaining after milling as a percentage of the total weight of the paddy. Breakage of grain during milling reduces the percentage of whole grain, and it can be due to a number of factors. Among grain quality traits, grain chalkiness is important since it affects milling, appearance, eating and cooking qualities. Chalk is the opaque area in the rice grain and is an undesirable trait. Chalky areas occur because of malformed starch granules with air spaces between them. The chalkiness is caused by suboptimal starch accumulation during the final stages of grain filling, causing a disordered cellular structure, rounder amyloplasts, and more air spaces. Chalky grains are more brittle than non-chalky grains and can break more easily during milling. The chalkiness negatively affects both the milling and cooking properties of rice and represents a major problem in many rice-producing areas of

the world. Chalk thus affects both the head rice yield (HYC), thereby decreasing the farm income and grain quality, decreasing the profits for farmers, and the marketability of rice.
[003] In recent years, with the increase of living standard, more and more attention has been paid on rice quality, including the appearance quality, processing quality, nutritional quality, cooking and eating quality, etc.
[004] US10485196B2 discloses plants with an improved organ phenotype. In particular, the invention relates to rice plants which have better grain quality and altered seed phenotype due to increased expression of GW7 (LOC_Os07g41200) or due to a combination of increased expression of GW7 and reduced expression of GS3 (OsSPL16).
[005] US 10260021B2 relates to rice oil, rice bran, and rice seeds which have altered levels of oleic acid, palmitic acid and/or linoleic acid. It provides methods for genetically modifying rice plants such that rice oil, rice bran and rice seeds produced therefrom have altered levels of oleic acid, palmitic acid and/or linoleic acid. [006] Although, several major or minor quantitative trait locus (QTLs) associated with grain chalkiness have been identified, and few genes that contribute to carbon flow or starch biosynthesis, per se, have also been identified to be associated with the chalkiness trait. However, there is still a dire need to develop a strategy that can eliminate the undesirable trait of chalky endosperm or white belly grains.
SUMMARY OF THE INVENTION
[007] In an aspect of the present disclosure, there is provided a recombinant DNA construct comprising a seed-specific promoter operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1.
[008] In another aspect of the present disclosure, there is provided a recombinant vector comprising the recombinant construct, said recombinant DNA construct comprising a seed-specific promoter operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1.

[009] In another aspect of the present disclosure, there is provided a recombinant host cell comprising the recombinant vector as described herein. [0010] In another aspect of the present disclosure, there is provided a transgenic plant or parts thereof having an improved grain quality comprises the recombinant construct, said recombinant DNA construct comprising a seed-specific promoter operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1.
[0011] In another aspect of the present disclosure, there is provided a method for obtaining a transgenic plant or parts thereof expressing a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, said method comprising: (a) obtaining a recombinant vector as described herein; (b) transforming a host cell with the recombinant vector to obtain a recombinant host cell; (c) transforming a plant cell with the recombinant host cell to obtain putative transformants; and (d) screening the putative transformants to obtain a transgenic plant or parts thereof expressing a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1. [0012] In another aspect of the present disclosure, there is provided a food product from the transgenic plant as described herein.
[0013] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0014] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0015] Figure 1A and Figure IB depicts immunolocalisation of OsMADS29 (M29) in developing endosperm and embryo, in accordance with an embodiment of the present disclosure.
[0016] Figure 1C depicts the expression domains of the two promoters used to drive the expression of M29, in accordance with an embodiment of the present disclosure. [0017] Figure 2 depicts the schematic representation of recombinant construct (gene expression constructs) used for modifying expression profile of M29 in rice. RB: right border, LB: left border, NosT: Nopaline synthase terminator, M29: OsMADS29 coding sequence, P-Oleosin-18: 01eosinl8 promoter, P-Nos: nopaline synthase promoter, FtPTII: hygromycin phosphotransferase gene, pAg7: agropine synthase gene terminator, P-Prolamin26: Prolamin 26 promoter, in accordance with an embodiment of the present disclosure.
[0018] Figure 3 depicts a graphical depiction of grain weight, length and width and expression of M29 as measured by Q-PCR. The left-hand side graph is for 01eosinl8:M29 (018:M29) transgenics and the right-hand side graph represents data for Prolamin26:M29 (P26:M29) transgenics, in accordance with an embodiment of the present disclosure.
[0019] Figure 4(A) depicts half-cut fully matured seed which is chalky in wild type Pusa Basmati (WT PB1), but is translucent in transgenics (Left panel (018:M29 transgenics)) and Figure 4(B) depicts the starch staining pattern with Lugol's stain where chalky endosperm belly was seen in WT and a translucent belly in seen in transgenics. Scale bar = 500 um and the (B) right hand side panel is for P26:M29 transgenics, in accordance with an embodiment of the present disclosure. [0020] Figure 5 depicts representative transmitted light images of wild type (WT), 018:M29 and P26:M29 transgenic seed where the opaque region shows chalkiness, in accordance with an embodiment of the present disclosure.
[0021] Figure 6 depicts percent decrease in chalkiness of the grains of 018:M29 and P26:M29 transgenics as compared to WT, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION

[0022] Those skilled in the art will be aware that the present disclosure is subject to
variations and modifications other than those specifically described. It is to be
understood that the present disclosure includes all such variations and modifications.
The disclosure also includes all such steps, features, compositions, and compounds
referred to or indicated in this specification, individually or collectively, and any and
all combinations of any or more of such steps or features.
[0023] Sequences listing description. Nucleic acid sequences listed in the
accompanying sequence listing and referenced herein are shown using standard letter
abbreviations for nucleotide bases. Only one strand of each nucleic acid sequence is
shown, but the complementary strand is understood to be included by any reference
to the displayed strand.
[0024] SEQ ID NO: 1 depicts the amino acid sequence of OsMADS29 (M29)
[0025] SEQ ID NO: 2 depicts the nucleotide sequence of OsMADS29 gene
[0026] SEQ ID NO: 3 depicts the nucleotide sequence of 01el8:M29_NosT
[0027] SEQ ID NO: 4 depicts the nucleotide sequence of Pro26: M29_NosT
[0028] SEQ ID NO: 5 depicts the nucleotide sequence of M29 forward primer
[0029] SEQ ID NO: 6 depicts the nucleotide sequence of M29 reverse primer
[0030] SEQ ID NO: 7 depicts the nucleotide sequence of Prolamin26 forward primer
[0031] SEQ ID NO: 8 depicts the nucleotide sequence of Prolamin26 reverse primer
[0032] SEQ ID NO: 9 depicts the nucleotide sequence of 01eosinl8 forward primer
[0033] SEQ ID NO: 10 depicts the nucleotide sequence of 01eosinl8 reverse primer
[0034] SEQ ID NO: 11 depicts the nucleotide sequence of 01eosinl8 specific
forward primer
[0035] SEQ ID NO: 12 depicts the nucleotide sequence of M29 gene specific reverse
primer
[0036] SEQ ID NO: 13 depicts the nucleotide sequence of Prolamin26 sequence
specific forward primer
[0037] SEQ ID NO: 14 depicts the nucleotide sequence of M29 qFP
[0038] SEQ ID NO: 15 depicts the nucleotide sequence of NosT RP
[0039] SEQ ID NO: 16 depicts the nucleotide sequence of 01eosinl8 (018)
promoter

[0040] SEQ ID NO: 17 depicts the nucleotide sequence of Prolamin 26 promoter
Definitions
[0041] For convenience, before further description of the present disclosure, certain
terms employed in the specification, and examples are delineated here. These
definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the meanings
recognized and known to those of skill in the art, however, for convenience and
completeness, particular terms and their meanings are set forth below.
[0042] The articles "a", "an" and "the" are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0043] The terms "comprise" and "comprising" are used in the inclusive, open sense,
meaning that additional elements may be included. It is not intended to be construed
as "consists of only".
[0044] Throughout this specification, unless the context requires otherwise the word
"comprise", and variations such as "comprises" and "comprising", will be
understood to imply the inclusion of a stated element or step or group of element or
steps but not the exclusion of any other element or step or group of element or steps.
[0045] The term "including" is used to mean "including but not limited to".
"Including" and "including but not limited to" are used interchangeably.
[0046] For the purposes of the present document, the term "recombinant" refers to
an artificial combination of two otherwise separated segments of sequences, e.g., by
chemical synthesis, or manipulation of isolated segments of nucleic acids by genetic
engineering techniques.
[0047] The term "recombinant construct" comprises an artificial combination of
nucleic acid fragments, e.g., regulatory and coding sequences that are not all found
together in nature. For example, a construct may comprise regulatory sequences and
coding sequences that are derived from different sources, or regulatory sequences
and coding sequences derived from the same source, but arranged in a manner
different than that found in nature. Such a construct may be used by itself or may be
used in conjunction with a vector. If a vector is used, then the choice of vector is

dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. In the present disclosure, "recombinant construct", "recombinant DNA construct", "gene expression construct", and "construct", are used interchangeably herein. [0048] As used herein, the term "transgenic plant" refers to a plant which comprises within its genome a heterologous polynucleotide introduced by a transformation step. The heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant construct. A transgenic plant can also comprise more than one heterologous polynucleotide within its genome. Each heterologous polynucleotide may confer a different trait to the transgenic plant. A heterologous polynucleotide can include a sequence that originates from a foreign species, or, if from the same species, can be substantially modified from its native form. Transgenic can include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The alterations of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods, by the genome editing procedure described herein that does not result in an insertion of a foreign polynucleotide, or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation are not intended to be regarded as transgenic. [0049] As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription, that of the 5' untranslated region (UTR) that is involved in recognition and binding of RNA polymerase and other regulatory proteins to initiate transcription.
[0050] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.
[0051] As used herein, the term "host cell" means a cell, which comprises a heterologous nucleic acid sequence of the disclosure, which contains a vector and

supports the replication and/or expression of the expression vector. According to the present disclosure, the host cell is selected from plant cell, or a bacterium. The preferred embodiments of bacterial cell: E. coli, Agrobacterium tumefaciens. The preferred embodiment of plant cell: rice.
[0052] The term "decrease in chalkiness" refers to a decrease in the opaqueness in a grain of a transgenic plant having the recombinant construct of the present disclosure as compared to that of the corresponding wild-type plant (non-transformed plant or control plant). In the wild-type plants the chalkiness is known to be caused by suboptimal starch accumulation.
[0053] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0055] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein. [0056] The undesirable trait of chalkiness affects milling, appearance, cooking qualities, and thereby represents major problems by decreasing the farm income, because of decreased marketability of rice, etc. While there are various agronomic and genetic methods, and compositions available in the art, none of the methods appears to be effective or practical in improving the grain quality in relation to chalkiness as the available strategies do not come as close to eliminating the

undesirable trait of chalky endosperm or white belly grains as described in the present disclosure.
[0057] Thus, in order to overcome the aforementioned problems, the present disclosure provides a solution by providing a recombinant construct comprising the transcription factor OsMADS29 (MADS29 or M29) gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, which is operably linked to the seed-specific promoters. The MADS29 (M29) gene is a major regulator of seed development in rice, wherein the expression of M29 gene is confined explicitly to sub-aleuronic cell layers of endosperm and cells in the embryo surrounding tissues (Fig 1A, IB). In rice varieties that exhibit chalkiness, it is endosperm that shows air spaces and irregularly shaped starch grains, characteristic of the chalk. Therefore, to overcome this limitation, the present disclosure provides the recombinant construct comprising the polypeptide (SEQ ID NO: 1) as described herein, which augments the starch biosynthesis machinery in the endosperm, wherein the polypeptide (SEQ ID NO: 1) encoded by M29 gene (SEQ ID NO: 2) is expressed under the control of two seed-specific promoters, namely Prolamin 26 (2062 bp; P26) and Oleosin 18 (1249 bp; 018).
[0058] The expression patterns of the two promoters in the seed are subtly different from that of the M29 (Figure 1). While the expression oiM29 is confined explicitly to sub-aleuronic cell layers of endosperm and in the cells surrounding the embryo, the promoter P26 drives the expression of downstream genes in the embryo and aleurone layer with weaker, evenly distributed expression in the inner portion of endosperm, and the promoter 018 drives the expression specifically in the aleurone layer and embryo of the seed. By modifying the expression profile of M29 gene under the control of these two promoters to other areas of the seed, the present disclosure reduces the chalkiness significantly and also increases seed weight, length, and width.
[0059] Thus, the property of the recombinant construct to modify the expression profile of M29 gene is used to transform the rice plants to obtain the transgenic plants, wherein the transgenic plants express the polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, and wherein the transgenic plants exhibit a

significant decrease in chalkiness and increase in the seed weight, length, and width, as compared to wild type plant. The present disclosure also provides a method of obtaining transgenic plants. Since the transgenic plants exhibit an improved quality trait, such as reduced chalkiness, increased seed weight, length, and width, a food product such as grain can be obtained with desired quality traits, which directly helps the farmers to earn profits and also increases the marketability of the crop. Overall, the present disclosure has a significantly high commercial value because it can positively cut down losses during the milling process and positively impact farmer's income.
[0060] In an embodiment of the present disclosure, there is provided a recombinant DNA construct comprising a seed-specific promoter operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence as set forth inSEQIDNO: 1.
[0061] In an embodiment of the present disclosure, there is provided a recombinant DNA construct comprising a seed-specific promoter operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, wherein the polynucleotide has a nucleotide as set forth in SEQ ID NO: 2.
[0062] In an embodiment of the present disclosure, there is provided a recombinant DNA construct comprising a seed-specific promoter operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, wherein the seed-specific promoter is selected from the group consisting of prolamin 26, oleosin 18, RAL2, RAL4, CAPIP, 1.3 kDa and 2.3 kDa glutelin B-l (GluB-1), GluB-2, GluB-4, 16 kDa prolamin, 26 kDa alpha globulin Glb-1, alanine aminotransferase (AlaAT), glutamate synthase (GOGAT), starch branching enzyme (SBE1), rice embryo globulin-2 (REG2), Glutelin A-l (GluA-1), GluA-2, GluA-3, GluB-3, GluB-5, GluC, soluble starch synthase 2-3 (OsSSII-3/), rice sucrose synthase 3 (RSUS3/) and aleurone layer-specific 1 (AL1/ anthranilate N-hydroxycinnamoyl/ benzoyltransferase. In another embodiment of the present disclosure, the seed-specific promoter is prolamin 26. In yet another embodiment of the present disclosure, the seed-specific promoter is oleosin 18.

I/We Claim:

1. A recombinant DNA construct comprising a seed-specific promoter operably linked to a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1.
2. The recombinant DNA construct as claimed in claim 1, wherein the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO: 2.
3. The recombinant DNA construct as claimed in claim 1, wherein the seed-specific promoter is selected from the group consisting of prolamin 26, oleosin 18, RAL2, RAL4, CAPIP, 1.3 kDaglutelinB-1 (GluB-1), 2.3 kDaglutelinB-1 (GluB-1), GluB-2, GluB-4, 16 kDa prolamin, 26 kDa alpha globulin Glb-1, alanine aminotransferase (AlaAT), glutamate synthase (GOGAT), starch branching enzyme (SBE1), rice embryo globulin-2 (REG2), P-conglycinin promoter, Glutelin A-l (GluA-1), GluA-2, GluA-3, GluB-3, GluB-5, GluC, soluble starch synthase 2-3 (OsSSII-3/), rice sucrose synthase 3 (RSUS3/), and aleurone layer-specific 1 (ALU) anthranilate N-hydroxycinnamoyl/benzoyltransferase.

4. A recombinant vector comprising the recombinant construct as claimed in anyone of the claims 1-3.
5. A recombinant host cell comprising the recombinant vector as claimed in claim 4.
6. The recombinant host cell as claimed in claim 5, wherein the host cell is selected from the group consisting of bacteria, and plant cells.
7. The recombinant host cell as claimed in claim 6, wherein the host cell is bacteria, and wherein the said bacteria is selected from the group consisting of E. coli, and Agrobacterium tumefaciens.
8. A transgenic plant or parts thereof having an improved grain quality comprises the recombinant construct as claimed in anyone of the claims 1-3.
9. The transgenic plant as claimed in claim 8, wherein the transgenic plant exhibits a characteristic selected from the group consisting of: (a) decrease in grain chalkiness; (b) increase in grain weight; (c) increase in grain length; and (d) increase in grain width.

10. The transgenic plant as claimed in claim 8 or 9, wherein the plant is selected from the group consisting of rice, wheat, maize, sorghum, oat, and brachypodium.
11. A method for obtaining a transgenic plant or parts thereof expressing a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1, said method comprising:
a) obtaining a recombinant vector as claimed in claim 4;
b) transforming a host cell with the recombinant vector to obtain a recombinant host cell;
c) transforming a plant cell with the recombinant host cell to obtain putative transformants; and
d) screening the putative transformants to obtain a transgenic plant or parts thereof
expressing a polypeptide having an amino acid sequence as set forth in SEQ ID NO:
1.
12. The method as claimed in claim 11, wherein transforming a plant cell is done by a method selected from the group consisting of an Agrobacterium-mediated transformation, particle bombardment, stable callus-PEG/ electroporation mediated transformation, floral dip method, microprojectile bombardment, and bioactive beads-mediated transformation.
13. The method as claimed in claim 11, wherein the screening is done by a method selected from the group consisting of PCR, blotting, and hybridization.
14. A food product from the transgenic plant as claimed in claim 8.
15. The food product as claimed in claim 14, wherein the food product is selected from the group consisting of grains.
16. The food product as claimed in claim 14 or 15, wherein the plant is selected from the group consisting of rice, wheat, maize, sorghum, oat, and brachypodium.