Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS FOR PRODUCING TRANSGENIC PLANTS
OVEREXPRESSING NON-SYMBIOTIC HEMOGLOBIN CLASS-1 GENE,
AND APPLICATIONS THEREOF
FIELD OF INVENTION
[001] The subject matter disclosed herein broadly relates to the field of
plant
molecular biology, and in particular relates to the development of early
yielding and
enhanced fruiting transgenic plant lines.
BACKGROUND OF THE INVENTION
[002]Tomatoes and tomato products are one of the most familiar consumables in
the
normal human diet. Quantitatively, they are the most consumed non-starchy
vegetable
and are the most significant source of dietary lycopene. Tomatoes serves as a
powerful
antioxidant that has greater bioavailability after cooking and processing. In
addition to
the specific benefits of tomato consumption, it helps individuals to increase
vegetable
intake, leading to improved overall eating patterns, and ultimately, better
health.
[003]As per the Monthly Report Tomato, January-2018 by the Ministry of
Agriculture
and farmers welfare (agricoop.nic.in/annual-report), the data for the yearly
tomato
prices shows unpredictable patterns, due to which the costing and availability
al so
suffers for the common people. Helgi library data shows that India is becoming
an
emerging consumer of tomatoes and its name is not even mentioned in the list
of tomato
exporters or largest producers worldwide. Such differences between the
production and
the consumption patterns of our country could be a matter of concern
considering the
price of the vegetable for the common people.
[004] Leshem et al. (Non-invasive photoacoustic spectroscopic determination of
relative endogenous nitric oxide and ethylene content stoichiometry during the
ripening of strawberries Fragaria anannasa (Duch.) and avocados Persea
americana
(Mill.), Journal of Experimental Botany, Volume 51, Issue 349, August 2000,
Pages
1471-1473, haps ://doi.org/10. 1093/j exbot/51.349.1471) have studied the
relationship
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between nitric oxide and ethylene pathways. In post-harvest strawberries and
avocados,
it was observed that endogenous nitric oxide levels indirectly governed the
fruit
senescence by regulating the ethylene pathways that lead to fruit ripening and
senescence. Although, extensive studies are coming up with similar results,
but there
still persists a need for such studies to solve problems for various
climacteric fruits in
regard to their low yield cost-effective prices.
[005] Despite the extensive research done in this field, the presently
available
techniques are not able to practically help the farmers producing crops in
terms of yield,
cost effectiveness and availability of such enhanced crops. Also, despite the
substantial
ongoing research on phytoglobins, hardly any reports are available on
expression
studies of phytoglobins in tomato plants. Thus, there is still a dire need in
the state of
art to develop rich tomato varieties, which could result in meeting the
present-day
consumption needs, alongside taking care of ease of availability of such crops
at
affordable prices.
SUMMARY OF THE INVENTION
[006] In an aspect of the present disclosure, there is provided a recombinant
construct
comprising a nucleic acid fragment operably linked to a heterologous promoter,
wherein the nucleic acid fragment encodes a polypeptide having an amino acid
sequence with at least 95% identity to the sequence as set forth in SEQ ID NO:
2.
[007] In another aspect of the present disclosure, there is provided a
recombinant
vector comprising the recombinant construct as described herein.
[008] In another aspect of the present disclosure, there is provided a
recombinant host
cell comprising the recombinant construct as described herein, or the
recombinant
vector as described herein.
[009] In another aspect of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with a recombinant vector comprising the recombinant construct as described
herein
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or with a recombinant host cell comprising the recombinant vector or the
recombinant
construct as described herein, to obtain transformed plant cells; and (b)
screening the
transformed plant cells to obtain a transgenic plant cell, wherein the
transgenic plant
cell produces the polypeptide having an amino acid sequence with at least 95%
identity
to the sequence as set forth in SEQ ID NO: 2.
[0010] In another aspect of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
a recombinant vector comprising the recombinant construct as described herein
or with
a recombinant host cell comprising the recombinant vector or the recombinant
construct as described herein, to obtain transgenic plant cells; and (b)
regenerating a
transgenic plant from the transgenic plant cell, wherein the transgenic plant
exhibits
altered fruit phenotype, and early ripening of the fruits relative to a non-
transgenic plant
of the same species.
[0011] In another aspect of the present disclosure, there is provided a method
for
obtaining a transgenic Solanum lycopersicum plant, said method comprising: a)
obtaining a recombinant vector comprising the recombinant construct as
described
herein or a recombinant host cell comprising the recombinant vector or the
recombinant
construct as described herein; b) transforming a Solon urn lycopersicum
explant with
the recombinant vector or with the recombinant host cell obtained in step (a),
to obtain
a transformed plant cell; and c) regenerating a transgenic Solanum
lycopersicum plant
from the transformed plant cell, wherein the transgenic Solant1171
lycopersicum plant
exhibits early ripening and altered fruit phenotype relative to a non-
transgenic Solanutn
lycopersicum plant.
[0012] In another aspect of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: a) obtaining a
recombinant
vector comprising a nucleic acid fragment operably linked to a heterologous
promoter,
wherein the nucleic acid fragment encodes a polypeptide having an amino acid
sequence with at least 95% identity to the sequence as set forth in SEQ ID NO:
2; b)
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introducing the recombinant vector obtained in step (a) into a plant cell, to
obtain
putative transformed plant cells; c) screening the putative transformants, to
obtain
transgenic plant cell; and d) regenerating the transgenic plant cell, for
obtaining a
transgenic plant, wherein the transgenic plant exhibits early ripening and
altered fruit
phenotype relative to a non-transgenic plant of the same species.
[0013] In another aspect of the present disclosure, there is provided a
transgenic plant
cell comprising a nucleic acid fragment encoding a polypeptide having an amino
acid
sequence with at least 95% identity to the sequence as set forth in SEQ ID NO:
2.
These and other features, aspects, and advantages of the present subject
matter will be
better understood with reference to the following description. 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.
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 la depicts the tissue culture grown transgenic tomato plants
from the
cotyledonary leaves of the explant; Figure lb depicts hardening of tissue
culture grown
transgenic tomato plants for 7 days; Figure lc depicts transgenic tomato
plants
transferred to the soil for further growth, in accordance with an embodiment
of the
present disclosure.
[0016] Figure 2a depicts the gel-electrophoresis results from the PCR reaction
done to
screen transgenic tomato plants by using BASTA primers specific to the bar
gene
sequence present in the pEarleyGate 201 vector, as used in the present
disclosure;
Figure 2b depicts real-time PCR results done to screen transgenic tomato
plants; Figure
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2c depicts western blot results done to screen transgenic tomato plants, in
accordance
with an embodiment of the present disclosure.
[0017] Figure 3a depicts nitric oxide (NO) levels as per the
diaminofluorescein-
FM (DAF-FM) fluorescence test results of wild type (WT) and sample transgenic
tomato plants; Figure 3b depicts quantified NO fluorescence intensity in the
WT and
the transgenic tomato plants; Figure 3c depicts gaseous NO levels measured by
the
gas-phase chemiluminescence in WT and transgenic tomato plants, in accordance
with
an embodiment of the present disclosure.
[0018] Figure 4 depicts the slightly hypoxic conditions of the fruits produced
from the
transgenic tomato plants, in accordance with an embodiment of the present
disclosure.
[0019] Figure 5 shows the increased number of cherry-like tomato fruits in
transgenic
tomato plants with reduced weight and diameter, in accordance with an
embodiment
of the present disclosure.
[0020] Figure 6a depicts regular fruit ripening in the wild type tomato plants
having
normal NO concentrations; Figure 6b depicts early fruit ripening in transgenic
tomato
plants having less NO concentrations from the resulting overexpression lines;
Figure
6c depicts the representative images of average number of fruits per bunch
from wild
type tomato plants, and Figure 6d depicts the representative images of average
number
of fruits per bunch from nsilb over expression tomato plants, in accordance
with an
embodiment of the present disclosure.
[0021] Figure 7 depicts the delayed ripening process in wild types in
comparison to
transgenics overexpressing nsHb, suggesting that NO is involved in inhibition
of fruit
ripening in tomato, in accordance with an embodiment of the present
disclosure.
[0022] Figure 8 depicts the pEarley Gate 201 expression recombinant vector map
comprising the nucleic acid fragment nsHb, having a nucleic acid sequence with
at
least 95% identity to the sequence as set forth in SEQ ID NO: 1, in accordance
with an
embodiment of the present disclosure.
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[0023] Figure 9 depicts the gas chromatography¨mass spectrometry (GC-MS)
analysis
used to detect metabolites of tomato fruits, in accordance with an embodiment
of the
present disclosure.
[0024] Figure 10 depicts the variation in the levels of sugars between WT and
nsHb
over expression tomato fruits, detected using untargeted metabolite analysis,
in
accordance with an embodiment of the present disclosure.
[0025] Figure 11 depicts the ICP-MS analysis of WT and tisHb over expression
tomato
fruits, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0026] 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.
Definitions
[0027] 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.
[0028] 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.
[0029] 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".
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[0030] 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.
[0031] The term "including" is used to mean "including but not limited to".
"Including" and "including but not limited to" are used interchangeably.
[0032] The term "at least one" used herein refers to one or more and thus
includes
individual components as well as mixtures/combinations.
[0033] The term "phytoglobins" refers to globular plant proteins classified
into
the globin superfamily, which contain a heme prosthetic group. Phytoglobins
are
ubiquitously distributed in plants as they have been identified in algae and
land plants,
including primitive bryophytes and evolved monocots and dicots.
[0034] As used herein, the term "recombinant construct" is a construct build
by
laboratory methods of genetic recombination to bring together genetic
materials from
multiple sources, creating sequences that would not otherwise be found in the
genome.
[0035] The term "vector", as used herein, refers to a DNA molecule used as a
vehicle
to artificially carry foreign genetic material into another cell, where it can
be replicated and/or expressed. The term "recombinant vector" refers to a
vector
containing foreign DNA sequence.
[0036] The term "transgenic plants" refers to a plant that is obtained by
transforming
the recombinant construct of recombinant vector of the present disclosure into
a wild-
type or a non-transgenic plant by artificially introducing the vector or the
construct
through genetic engineering techniques.
[0037] The term "transgenic Solanurn lycopersicurn plant" or "transgenic
tomato
plants" refers to the nsHb over expressing tomato plants that are obtained by
transforming a wild-type or a non-transgenic plant by artificially introducing
the vector
or the construct through genetic engineering techniques as per the present
disclosure.
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[0038] The term "transformation" refers to a process involving genetic
alteration of a
plant cell resulting from the direct uptake and incorporation of exogenous
genetic
material from its surroundings through the cell membrane.
[0039] The term "heterologous" refers to a transfected DNA part, that is not
of the
host's autonomous DNA or simply isn't a part of the host's genetic material.
The term
"heterologous promoter" used herein refers to the constitutive promoter
obtained from
the other source than the host per se.
[0040] The term "OE" in the examples refer to the nsHb "over-expression" lines
of
transgenic tomato developed herein, as per the present disclosure.
Sequences used in the present disclosure:
[0041] SEQ ID NO: 1 depicts the nucleic acid sequence encoding non-symbiotic
hemoglobin.
ATGAGTAGCTTTAGTGAAGAACAAGAAGCTTTGGTAGTCAAATCATGGGG
GTCTATGAAGAAGGATGCTGGTGAATGGGGTCTCAAATTCTTTCTCAAGA
TATTTGAAATAGCACCATCAGCAAAAAAGATGTTCTCATTCCTCAAAGAT
TCAAATGTGCCATTGGATCAAAATCCTAAGCTCAAGATTCATGCTAAATC
TGTCCTTGTTATGACTTGTGAAGCGGCAGTTCAACTTAGAAAAGCTGGAA
AGGTTGTGGTGAGGGATTCCACTCTCAAAAAAATAGGTGCTACACACTTT
AAGTATGGTGTGGTTGATGAGCACTTTGAGGTAACAAAATATGCCTTGTT
GGAGACAATAAAAGAAGCAAGTCAAGAAATGTGGAGTGTTGAGATGAAG
AATGCATGGGGAGAGGCCTATGATCAATTGGTTAGTGCTATCAAGACTGA
GAT GAAGTAG
[0042] SEQ ID NO: 2 depicts the amino acid sequence of non-symbiotic
hemoglobin
class 1 gene (nsHb1).
>NP_001234498.1 non-symbiotic hemoglobin class 1 [Solunum lycopenicum]
MSSFSEEQEALVVKSWGSMKKDAGEWGLKFFLKIFEIAPSAKKMFSFLKDSN
VPLDQNPKLKIHAKSVLVMTCEAAVQLRKAGKVVVRDSTLKKIGATHFKYG
VVDEHFEVTKYALLETIKEAS QEMWSVEMKNAWGEAYDQLVSAIKTEMK
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[0043] SEQ ID NO: 3 depicts the forward primer nucleic acid sequence used for
amplification of Solanum lycopersicum non-symbiotic hemoglobin class 1 gene
(SlIVSH1).
CACCATGAGTAGCTTTAGTGAAGA
[0044] SEQ ID NO: 4 depicts the reverse prhner nucleic acid sequence used for
amplification of Solon urn lycopersicum non-symbiotic hemoglobin class 1 gene
(SlAISH1).
TAGCACACAAATTAGATTAT
[0045] 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.
[0046] As discussed in the background section of the present disclosure, the
ratio
between the consumption and production of tomato in India is not favorable for
both
farmers and consumers because being a highly disease susceptible variety,
tomato face
hurdles to successfully reaching the commercialization platform. Limited
fruiting and
the cropping cycles also hampers the process of mass production. Although,
advanced
tomato varieties have been developed to resist major problems including both
abiotic
and biotic stresses, but there still persists a dearth in further enhancing
the tomato
production to meet the current requirements and solving problems of ever-
increasing
prices of such basic vegetable.
[0047] In order to address the aforementioned limitations, the present
disclosure
discloses a method for obtaining a transgenic Solarium lycopersicum plant,
developed
through Agrobacterium mediated transformation. The transgenic lines involve
the
overexpression of phytoglobin gene nsHb (said gene encoding a polypeptide
having an
amino acid sequence having at least 95% identity to SEQ ID NO: 2) resulting in
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reduced nitric oxide (NO) levels, as phytoglobin 1 functionally scavenges
nitric oxide
which plays a role in regulating ethylene levels in fruits. On overexpression
of
phytoglobin gene, the produced transgenic plant shows increased number of
fruits per
plant as compared to wild type Solarium lycopersicum (tomato) plant. The
transgenic
plants developed from the method as disclosed herein also shows accelerated
fruit
setting time and the fruits obtained are of cherry-like appearance. As per the
disclosure,
the increased fruit number from the developed transgenic lines can enhance
tomato
production to satisfy consumption needs. Shortened time period for fruit
ripening can
also help in producing multiple crops every year from the view of urban
agricultural
form. Enhanced yield from the present transgenic lines will also help to
negotiate the
damages or spoilage incurred to the total yield due to disease/infection or
transportation. Early ripening of fruits will also help in reducing the use of
artificial
ripening agents that are generally used in case of tomatoes. Cherry like
tomatoes are
comparatively smaller than the wild type tomatoes, which saves the tomatoes
from
getting fruit cracks due to their smaller sizes, minimizing the additional
yield damages.
The relatively smaller sized tomatoes developed from the transgenic plants by
the
method disclosed herein are uniform in shape, which can also help in easy
transportation and marketing in small packets with minimal losses. Further,
the
transgenic tomato plant as per the present disclosure produces tomato which
have
cherry-like in appearance, thereby, providing tomatoes that have high value in
terms of
their sensorial attributes and are a requirement in culinary decoration and as
an
ingredient in many dishes. The present disclosure thus provides a recombinant
construct comprising a nucleic acid sequence as set forth in SEQ ID NO: 1
linked to a
heterologous promoter. The present disclosure further discloses a recombinant
vector
comprising the recombinant construct as described herein. The heterologous
promoter
disclosed herein is not limited to single CaMV promoter, it extends to CaMV
promoter
and double 35S promoter. A bacterial recombinant host cell is disclosed which
comprises either a recombinant construct or a recombinant vector as disclosed
herein.
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The present disclosure further discloses a method of obtaining a transgenic
plant cell
that is developed by transforming an explant with either a recombinant vector
or by a
recombinant host cell to obtain a transformed plant cell, transformed plant
cells are
further screened for the transgenic plant cells , which produces the
polypeptide
sequence having amino acid sequence with at least 95% identity to the sequence
as set
forth in SEQ ID NO: 2. A method for obtaining a transgenic plant is further
disclosed,
which comprises transforming an explant with a recombinant vector or a
recombinant
host cell as disclosed herein to obtain a transgenic plant cell, followed by
regenerating
obtained transgenic cells to get transgenic plants which exhibit altered fruit
phenotype,
and early ripening of the fruits relative to a non-transgenic plant of the
same species.
The transgenic tomato plants also exhibit enhanced nutritional properties by
enhancement of certain elements such as Myo-Inositol, Na, Mg, K and Ca, as
compared
to wild type tomato plants. Further, a higher accumulation of sugars such as
glucose,
sucrose, and fructose are also observed in transgenic tomato plants as
compared to wild
type tomato plants. Hence, owing to the enhanced nutritional properties,
transgenic
tomato plants is suitable for human consumption and can also be deployed in a
food
product.
[0048] The present disclosure also discloses a method for obtaining a
transgenic
tomato plant in particular, said method comprises, obtaining a recombinant
vector or a
recombinant host cell as described herein, to transform the tomato explant to
get
transformed plant cell. The transformed plant cell is subjected to
regeneration to obtain
a transgenic tomato plant showing early ripening and altered fruit phenotype
relative
to a non-transgenic tomato plant. As per the present disclosure, the
transgenic plants
also exhibit increase in fruit number and decrease in fruit weight relative to
non-
transgenic plants. The method of transformation as per the present disclosure
is not
limited to Agrobacterium mediated transformation, but also extends to particle
gun
bombardment method, in-planta transformation method, liposome mediated
transformation method, protoplast transformation method, microinjection, and
macro
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injection method. Also, methods of producing transgenic plant cells or plants
per se is
not restricted to tomato. The present disclosure extends further to production
of other
climacteric plants such as banana, mango, sugar apple, peach, apricot and the
like.
[0049] 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.
[0050] In an embodiment of the present disclosure, there is provided a
recombinant
construct comprising a nucleic acid fragment operably linked to a heterologous
promoter, wherein the nucleic acid fragment encodes a polypeptide having an
amino
acid sequence with at least 95% identity to the sequence as set forth in SEQ
ID NO: 2.
In another embodiment of the present disclosure, the nucleic acid fragment
encodes a
polypeptide having an amino acid sequence with at least 95.5%, or 96%, or
96.5%, or
97%, or 97.5%, or 98%, or 98.5%, or 99%, or 99.5%, or 99.9% identity to the
sequence
as set forth in SEQ ID NO: 2.
[0051] In an embodiment of the present disclosure, there is provided a
recombinant
construct comprising a nucleic acid fragment operably linked to a heterologous
promoter, wherein the nucleic acid fragment encodes a polypeptide having an
amino
acid sequence with at least 95% identity to the sequence as set forth in SEQ
ID NO: 2,
In another embodiment of the present disclosure, the nucleic acid fragment
encodes a
polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2.
[0052] In an embodiment of the present disclosure, there is provided a
recombinant
construct comprising a nucleic acid fragment operably linked to a heterologous
promoter, wherein the nucleic acid fragment encodes a polypeptide having an
amino
acid sequence with at least 95% identity to the sequence as set forth in SEQ
ID NO: 2,
and wherein the nucleic acid fragment is having a nucleic acid sequence with
at least
95% identity to the sequence as set forth in SEQ ID NO: 1. In another
embodiment, the
nucleic acid fragment is having a nucleic acid sequence with at least 95.5%,
or 96%,
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or 96.5%, or 97%, or 97.5%, or 98%, or 98.5%, or 99%, or 99.5% identity to the
sequence as set forth in SEQ ID NO: 1.
[0053] In an embodiment of the present disclosure, there is provided a
recombinant
construct comprising a nucleic acid fragment operably linked to a heterologous
promoter, wherein the nucleic acid fragment encodes a polypeptide having an
amino
acid sequence with at least 95% identity to the sequence as set forth in SEQ
ID NO: 2,
and wherein the nucleic acid encodes a polypeptide having an amino acid
sequence as
set forth in SEQ ID NO: 2, and wherein the nucleic acid fragment is having a
nucleic
acid sequence as set forth in SEQ ID NO: 1.
[0054] In an embodiment of the present disclosure, there is provided a
recombinant
construct comprising a nucleic acid fragment operably linked to a heterologous
promoter, wherein the nucleic acid fragment encodes a polypeptide having an
amino
acid sequence as set forth in SEQ ID NO: 2.
[0055] In an embodiment of the present disclosure, there is provided a
recombinant
construct comprising a nucleic acid fragment operably linked to a heterologous
promoter, wherein the nucleic acid fragment encodes a polypeptide having an
amino
acid sequence as set forth in SEQ ID NO: 2, and wherein the nucleic acid
fragment is
having a nucleic acid sequence as set forth in SEQ ID NO: I.
[0056] In an embodiment of the present disclosure, there is provided a
recombinant
construct comprising a nucleic acid fragment operably linked to a heterologous
promoter, wherein the nucleic acid fragment encodes a polypeptide having an
amino
acid sequence with at least 95% identity to the sequence as set forth in SEQ
ID NO: 2,
and wherein the heterologous promoter is selected from the group consisting of
CaMV
promoter, double 35 S promoter, a single 35 S promoter. In another embodiment
of the
present disclosure, the heterologous promoter is CaMV promoter. In yet another
embodiment of the present disclosure, the heterologous promoter is double 35 S
promoter. In one another embodiment of the present disclosure, the
heterologous
promoter is a single 35 S promoter.
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[0057] In an embodiment of the present disclosure, there is provided a
recombinant
construct comprising a nucleic acid fragment operably linked to a heterologous
promoter having an amino acid sequence as set forth in SEQ ID NO: 2, and
wherein
the heterologous promoter is selected from the group consisting of CaMV
promoter,
double 35 S promoter.
[0058] In an embodiment of the present disclosure, there is provided a
recombinant
vector comprising a recombinant construct, said recombinant construct
comprising a
nucleic acid fragment operably linked to a heterologous promoter, wherein the
nucleic
acid fragment encodes a polypeptide having an amino acid sequence with at
least 95%
identity to the sequence as set forth in SEQ ID NO: 2.
[0059] In an embodiment of the present disclosure, there is provided a
recombinant
vector comprising a recombinant construct, said recombinant construct
comprising a
nucleic acid fragment operably linked to a heterologous promoter, wherein the
nucleic
acid fragment encodes a polypeptide having an amino acid sequence as set forth
in
SEQ ID NO: 2.
[0060] In an embodiment of the present disclosure, there is provided a
recombinant
vector comprising a recombinant construct, said recombinant construct
comprising a
nucleic acid fragment operably linked to a heterologous promoter, wherein the
nucleic
acid fragment encodes a polypeptide having an amino acid sequence with at
least 95%
identity to the sequence as set forth in SEQ ID NO: 2, and wherein the nucleic
acid
fragment is having a nucleic acid sequence with at least 95% identity to the
sequence
as set forth in SEQ ID NO: 1.
[0061] In an embodiment of the present disclosure, there is provided a
recombinant
vector comprising a recombinant construct, said recombinant construct
comprising a
nucleic acid fragment operably linked to a heterologous promoter, wherein the
nucleic
acid fragment encodes a polypeptide having an amino acid sequence as set forth
in
SEQ ID NO: 2, and wherein the nucleic acid fragment is having a nucleic acid
sequence
as set forth in SEQ ID NO: 1.
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[0062] In an embodiment of the present disclosure, there is provided a
recombinant
vector comprising a recombinant construct, said recombinant construct
comprising a
nucleic acid fragment operably linked to a heterologous promoter, wherein the
nucleic
acid fragment encodes a polypeptide having an amino acid sequence with at
least 95%
identity to the sequence as set forth in SEQ ID NO: 2, and wherein the
heterologous
promoter is selected from the group consisting of CaMV promoter, double 35 S
promoter, and single 35 S promoter.
[0063] In an embodiment of the present disclosure, there is provided a
recombinant
host cell comprising a recombinant vector, said recombinant vector comprising
a
recombinant construct, as described herein.
[0064] In an embodiment of the present disclosure, there is provided a
recombinant
host cell comprising a recombinant construct, said recombinant construct
comprising a
nucleic acid fragment operably linked to a heterologous promoter, wherein the
nucleic
acid fragment encodes a polypeptide having an amino acid sequence with at
least 95%
identity to the sequence as set forth in SEQ ID NO: 2.
[0065] In an embodiment of the present disclosure, there is provided a
recombinant
host cell comprising a recombinant construct, said recombinant construct
comprising a
nucleic acid fragment operably linked to a heterologous promoter, wherein the
nucleic
acid fragment encodes a polypeptide having an amino acid sequence as set forth
in
SEQ ID NO: 2.
[0066] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
expl ant
with the recombinant vector as described herein, to obtain transformed plant
cells; and
(b) screening the transformed plant cells to obtain a transgenic plant cell,
wherein the
transgenic plant cell produces the polypeptide having an amino acid sequence
with at
least 95% identity to the sequence as set forth in SEQ ID NO: 2.
[0067] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
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with the recombinant vector as described herein, to obtain transformed plant
cells; and
(b) screening the transformed plant cells to obtain a transgenic plant cell,
wherein the
transgenic plant cell produces the polypeptide having an amino acid sequence
as set
forth in SEQ ID NO: 2.
[0068] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant vector as described herein, to obtain transformed plant
cells; and
(b) screening the transformed plant cells to obtain a transgenic plant cell,
wherein the
transgenic plant cell produces the polypeptide having an amino acid sequence
as set
forth in SEQ ID NO: 2, and wherein the nucleic acid fragment is having a
nucleic acid
sequence as set forth in SEQ ID NO: 1.
[0069] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant vector as described herein, to obtain transformed plant
cells; and
(b) screening the transformed plant cells to obtain a transgenic plant cell,
wherein the
transgenic plant cell produces the polypeptide having an amino acid sequence
with at
least 95% identity to the sequence as set forth in SEQ ID NO: 2, and wherein
the nucleic
acid fragment is having a nucleic acid sequence with at least 95% identity to
the
sequence as set forth in SEQ ID NO: 1_
[0070] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant vector as described herein, to obtain transformed plant
cells; and
(b) screening the transformed plant cells to obtain a transgenic plant cell,
wherein the
transgenic plant cell produces the polypeptide having an amino acid sequence
as set
forth in SEQ ID NO: 2, and wherein the nucleic acid fragment is having a
nucleic acid
sequence as set forth in SEQ ID NO: I.
[0071] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
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with the recombinant host cell as described herein, to obtain transformed
plant cells;
and (b) screening the transformed plant cells to obtain a transgenic plant
cell, wherein
the transgenic plant cell produces the polypeptide having an amino acid
sequence with
at least 95% identity to the sequence as set forth in SEQ ID NO: 2.
[0072] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant host cell as described herein, to obtain transformed
plant cells;
and (11) screening the transformed plant cells to obtain a transgenic plant
cell, wherein
the transgenic plant cell produces the polypeptide having an amino acid
sequence as
set forth in SEQ ID NO: 2.
[0073] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant host cell as described herein, to obtain transformed
plant cells;
and (b) screening the transformed plant cells to obtain a transgenic plant
cell, wherein
the transgenic plant cell produces the polypeptide having an amino acid
sequence with
at least 95% identity to the sequence as set forth in SEQ ID NO: 2, and
wherein the
nucleic acid fragment is having a nucleic acid sequence with at least 95%
identity to
the sequence as set forth in SEQ ID NO: L
[0074] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant host cell as described herein, to obtain transformed
plant cells;
and (11) screening the transformed plant cells to obtain a transgenic plant
cell, wherein
the transgenic plant cell produces the polypeptide having an amino acid
sequence with
at least 95% identity to the sequence as set forth in SEQ ID NO: 2, and
wherein the
nucleic acid fragment is having a nucleic acid sequence with at least 95%
identity to
the sequence as set forth in SEQ ID NO: 1, and wherein the nucleic acid
encodes a
polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, and
wherein
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the nucleic acid fragment is having a nucleic acid sequence as set forth in
SEQ ID NO:
1.
[0075] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant host cell as described herein, to obtain transformed
plant cells;
and (b) screening the transformed plant cells to obtain a transgenic plant
cell, wherein
the transgenic plant cell produces the polypeptide having an amino acid
sequence as
set forth in SEQ ID NO: 2, and wherein the nucleic acid fragment is having a
nucleic
acid sequence as set forth in SEQ ID NO: 1.
[0076] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant construct as described herein, to obtain transformed
plant cells;
and (b) screening the transformed plant cells to obtain a transgenic plant
cell, wherein
the transgenic plant cell produces the polypeptide having an amino acid
sequence with
at least 95% identity to the sequence as set forth in SEQ ID NO: 2.
[0077] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant construct as described herein, to obtain transformed
plant cells;
and (b) screening the transformed plant cells to obtain a transgenic plant
cell, wherein
the transgenic plant cell produces the polypeptide having an amino acid
sequence as
set forth in SEQ ID NO: 2.
[0078] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell, said method comprising: (a) transforming an
explant
with the recombinant construct as described herein, to obtain transformed
plant cells;
and (b) screening the transformed plant cells to obtain a transgenic plant
cell, wherein
the transgenic plant cell produces the polypeptide having an amino acid
sequence as
set forth in SEQ ID NO: 2, and wherein the nucleic acid fragment is having a
nucleic
acid sequence as set forth in SEQ ID NO: 1.
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[0079] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant cell as described herein, wherein the transgenic
plant cell
is Solanum lycopersicum cell.
[0080] In an embodiment of the present disclosure, there is provided a method
for
obtaining a Lransgenic plant, said method comprising: (a) transforming an
explant with
the recombinant vector as described herein, to obtain transgenic plant cells;
and (b)
regenerating a transgenic plant from the transgenic plant cell, wherein the
transgenic
plant exhibits altered fruit phenotype, and early ripening of the fruits
relative to a non-
transgenic plant of the same species.
[0081] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
the recombinant host cell as described herein, to obtain transgenic plant
cells; and (b)
regenerating a transgenic plant from the transgenic plant cell, wherein the
transgenic
plant exhibits altered fruit phenotype, and early ripening of the fruits
relative to a non-
transgenic plant of the same species.
[0082] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
the recombinant vector as described herein, to obtain transgenic plant cells;
and (b)
regenerating a transgenic plant from the transgenic plant cell, wherein the
transgenic
plant exhibits altered fruit phenotype, and early ripening of the fruits
relative to a non-
transgenic plant of the same species, and wherein transforming is done by a
method
selected from the group consisting of Agrobacterium mediated transformation
method,
particle gun bombardment method, in-planta transformation method, liposome
mediated transformation method, protoplast transformation method,
microinjection,
and macro injection. In another embodiment of the present disclosure,
transforming is
done by Agrobacterium mediated transformation method.
[0083] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
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the recombinant host cell as described herein, to obtain transgenic plant
cells; and (b)
regenerating a transgenic plant from the transgenic plant cell, wherein the
transgenic
plant exhibits altered fruit phenotype, and early ripening of the fruits
relative to a non-
transgenic plant of the same species, and wherein transforming is done by a
method
selected from the group consisting of Agrobucterium mediated transformation
method,
particle gun bombardment method, in-planta transformation method, liposome
mediated transformation method, protoplast transformation method,
microinjection,
and macro injection.
[0084] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
the recombinant vector as described herein, to obtain transgenic plant cells;
and (b)
regenerating a transgenic plant from the transgenic plant cell, wherein the
transgenic
plant exhibits altered fruit phenotype, and early ripening of the fruits
relative to a non-
transgenic plant of the same species, and wherein the transgenic plant is
Solanum
lycopersicum.
[0085] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
the recombinant vector as described herein , to obtain transgenic plant cells;
and (b)
regenerating a transgenic plant from the transgenic plant cell, wherein the
transgenic
plant exhibits altered fruit phenotype, and early ripening of the fruits
relative to a non-
transgenic plant of the same species, and wherein the transgenic plant is
Solunum
lycopersicum.
[0086] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
the recombinant host cell as described herein, to obtain transgenic plant
cells; and (b)
regenerating a transgenic plant from the transgenic plant cell, wherein the
transgenic
plant exhibits altered fruit phenotype, and early ripening of the fruits
relative to a non-
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transgenic plant of the same species, and wherein the transgenic plant is
Solanum
lycopersicum.
[0087] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
the recombinant vector as described herein, to obtain transgenic plant cells;
and (b)
regenerating a transgenic plant from the transgenic plant cell, wherein the
transgenic
plant exhibits altered fruit phenotype, and early ripening of the fruits
relative to a non-
transgenic plant of the same species, and wherein the transgenic plant
exhibits an
increase in fruit numbers relative to a non-transgenic plant of the same
species.
[0088] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
the recombinant vector comprising the recombinant construct further comprising
a
nucleic acid fragment operably linked to a heterologous promoter, wherein the
nucleic
acid fragment encodes a polypeptide having an amino acid sequence as set forth
in
SEQ ID NO: 2, to obtain transgenic plant cells; and (b) regenerating a
transgenic plant
from the transgenic plant cell, wherein the transgenic plant exhibits altered
fruit
phenotype, and early ripening of the fruits relative to a non-transgenic plant
of the same
species.
[0089] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
the recombinant vector as described herein or with the recombinant host cell
comprising the recombinant vector further comprising the recombinant construct
further comprising a nucleic acid fragment operably linked to a heterologous
promoter,
wherein the nucleic acid fragment encodes a polypeptide having an amino acid
sequence with at least 95% identity to the sequence as set forth in SEQ ID NO:
2, and
wherein recombinant construct comprises of the nucleic acid that encodes a
polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2 or the
recombinant construct comprising a nucleic acid fragment operably linked to a
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heterologous promoter, wherein the nucleic acid fragment encodes a polypeptide
having an amino acid sequence with at least 95% identity to the sequence as
set forth
in SEQ ID NO: 2, and wherein recombinant construct comprises of the nucleic
acid
that encodes a polypeptide having an amino acid sequence as set forth in SEQ
ID NO:
2, and wherein the recombinant host cell is a bacterium, to obtain transgenic
plant cells;
and (b) regenerating a transgenic plant from the transgenic plant cell,
wherein the
transgenic plant exhibits altered fruit phenotype, and early ripening of the
fruits relative
to a non-transgenic plant of the same species.
[0090] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic plant, said method comprising: (a) transforming an
explant with
the recombinant vector as described herein or with the recombinant host cell
as
described herein, to obtain transgenic plant cells; and (b) regenerating a
transgenic
plant from the transgenic plant cell, wherein the transgenic plant exhibits
altered fruit
phenotype, and early ripening of the fruits relative to a non-transgenic plant
of the same
species, wherein the transgenic plant is selected from the group consisting of
tomato,
banana, mango, sugar apple, peach, apricot. In another embodiment of the
present
disclosure, the transgenic plant is tomato.
[0091] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic Solanum lycopersicum plant, said method comprising: a)
obtaining the recombinant vector as described herein; b) transforming a
Solanurn
lycopersicurn explant with the recombinant vector obtained in step (a), to
obtain a
transformed plant cell; and c) regenerating a transgenic Solanum lycopersicum
plant
from the transformed plant cell, wherein the transgenic Solanum lycopersicum
plant
exhibits early ripening and altered fruit phenotype relative to a non-
transgenic Solanurn
lycoper,s'icum plant.
[0092] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic Solanum lycopersicum plant, said method comprising: a)
obtaining the recombinant vector as described herein; b) transforming a
Solanum
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lycopersicum explant with the recombinant vector obtained in step (a), to
obtain a
transformed plant cell; and c) regenerating a transgenic Solanurn
lycopersicurn plant
from the transformed plant cell, wherein the transgenic Solanurn lycopersicurn
plant
exhibits early ripening and altered fruit phenotype relative to a non-
transgenic Solanum
lycopersicum plant.
[00931 In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic Solanum lycopersicum plant, said method comprising: a)
obtaining the recombinant vector as described herein; 11) transforming a
Solanum
lycopersicunz explant with the recombinant vector obtained in step (a), to
obtain a
transformed plant cell; and c) regenerating a transgenic Solanum lycopersicum
plant
from the transformed plant cell, wherein the transgenic Solanum lycopersicum
plant
exhibits early ripening and altered fruit phenotype relative to a non-
transgenic Solarium
lycopersicum plant, and wherein transgenic Solarium lycopersicum plant
produces the
polypeptide having an amino acid sequence with at least 95% identity to the
sequence
as set forth in SEQ ID NO: 2.
[0094] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic Solantern lycopersicum plant, said method comprising:
a)
obtaining the recombinant vector as described herein; b) transforming a
Solanum
lycopersicum explant with the recombinant vector obtained in step (a), to
obtain a
transformed plant cell; and c) regenerating a transgenic Sola num lycopersicum
plant
from the transformed plant cell, wherein the transgenic Solallt1171
lycopersicum plant
exhibits early ripening and altered fruit phenotype relative to a non-
transgenic Solanum
lycopersicum plant, and wherein transgenic Solanurn lycopersicum plant
produces the
polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2.
[0095] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic Solarium lycopersicum plant, said method comprising: a)
obtaining the recombinant vector as described herein; b) transforming a
Solanum
lycopersicum explant with the recombinant vector obtained in step (a), to
obtain a
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transformed plant cell; and c) regenerating a transgenic Solanum lycopersicurn
plant
from the transformed plant cell, wherein the transgenic Solanurn lycopersicurn
plant
exhibits early ripening and altered fruit phenotype relative to a non-
transgenic Solan urn
lycopersicurn plant, and wherein transgenic Solanurn lycopersicum plant
produces the
polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, and
wherein
the polypeptide is encoded by a nucleic acid fragment having a nucleic acid
sequence
as set forth in SEQ ID NO: L.
[0096] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic Solarium lycopersicurn plant, said method comprising:
a)
obtaining the recombinant host cell as described herein; b) transforming a
Solanum
lycopersicurn explant with the recombinant host cell obtained in step (a), to
obtain a
transformed plant cell; and c) regenerating a transgenic Solarium lycopersicum
plant
from the transformed plant cell, wherein the transgenic Solanum lycopersicurn
plant
exhibits early ripening and altered fruit phenotype relative to a non-
transgenic Solanum
lycopersicum plant.
[0097] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic Solanum lycopersicum plant as described herein, wherein
the
transgenic Solan urn lycopersicum plant exhibits an increase in fruit numbers
relative to
a non-transgenic plant of the same species, and wherein the transgenic plant
exhibits a
decrease in fruit weight relative to a non-transgenic plant of the same
species.
[0098] In an embodiment of the present disclosure, there is provided a method
for
obtaining a transgenic Solarium lycopersicum plant, said method comprising: a)
obtaining the recombinant host cell as described herein; b) transforming a
Solanum
lycopersicum explant with the recombinant host cell obtained in step (a), to
obtain a
transformed plant cell; and c) regenerating a transgenic Solarium lycopersicum
plant
from the transformed plant cell, wherein the transgenic Solanum lycopersicurn
plant
exhibits early ripening and altered fruit phenotype relative to a non-
transgenic Solarium
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lycopersicum plant, wherein transgenic Solanum lycopersicum plant produces the
polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2.
[0099] In an embodiment of the present disclosure, there is provided a
transgenic plant
obtained by the methods as described herein, wherein the transgenic plant
exhibits early
ripening and altered fruit phenotype relative to a non-transgenic plant of the
same
species. In another embodiment, the nucleic acid fragment encodes a
polypeptide
having an amino acid sequence with at least 95_5%, or 96%, or 96.5%, or 97%,
or
97.5%, or 98%, or 98.5%, or 99%, or 99.5%, or 99.9% identity to the sequence
as set
forth in SEQ ID NO: 2.
[00100] In an embodiment of the present disclosure, there is provided a
transgenic plant
comprising a nucleic acid fragment encoding a polypeptide having an amino acid
sequence as set forth in SEQ ID NO: 2.
[00101] In an embodiment of the present disclosure, there is provided a
transgenic plant
comprising a nucleic acid fragment encoding a polypeptide having an amino acid
sequence as set forth in SEQ ID NO: 2, wherein the nucleic acid fragment has a
nucleic
acid sequence as set forth in SEQ ID NO: 1.
[00102] In an embodiment of the present disclosure, there is provided a
transgenic plant
as described herein, wherein the plant is selected from the group consisting
of tomato,
banana, mango, sugar apple, peach, apricot. In another embodiment of the
present
disclosure, the plant is tomato.
[00103] In an embodiment of the present disclosure, there is provided a
transgenic plant
as described herein, wherein the transgenic plant exhibits early ripening and
altered
fruit phenotype relative to a non-transgenic plant of the same species.
[00104] In an embodiment of the present disclosure, there is provided a
transgenic plant
as described herein, wherein the transgenic plant exhibits an increase in
fruit numbers
relative to a non-transgenic plant of the same species, and wherein the
transgenic plant
exhibits a decrease in fruit weight relative to a non-transgenic plant of the
same species.
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[00105] In an embodiment of the present disclosure, there is provided a
transgenic plant
as described herein, wherein the transgenic plant exhibits early ripening and
altered
fruit phenotype relative to a non-transgenic plant of the same species,
wherein the
transgenic plant exhibits an increase in fruit numbers relative to a non-
transgenic plant
of the same species, and wherein the transgenic plant exhibits a decrease in
fruit weight
relative to a non-transgenic plant of the same species.
[00106] In an embodiment of the present disclosure, there is provided a
transgenic plant
as described herein, wherein the transgenic plant exhibits enhanced
nutritional
properties relative to a non-transgenic plant.
[00107] In an embodiment of the present disclosure, there is a food product
comprising
the transgenic plant, wherein the transgenic plant produces the polypeptide
having an
amino acid sequence as set forth in SEQ ID NO: 2.
[00108] Although the subject matter has been described in considerable detail
with
reference to certain embodiments thereof, other embodiments are possible. As
such,
the spirit and scope of the disclosure should not be limited to the
description of the
embodiments contained herein.
EXAMPLES
[00109] The disclosure will now be illustrated with the working examples,
which is
intended to illustrate the working of disclosure and not intended to take
restrictively to
imply any limitations on the scope of the present disclosure. Unless defined
otherwise,
all technical and scientific terms used herein have the same meaning as
commonly
understood to one ordinary person skilled in the art to which this disclosure
belongs.
Although methods and materials similar or equivalent to those described herein
can be
used in the practice of the disclosed methods and compositions, the exemplary
methods, devices and materials are described herein. It is to be understood
that this
disclosure is not limited to particular methods, and experimental conditions
described,
as such methods and conditions may apply.
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Materials and Methods
[00110] For the purpose of the present disclosure, Tomato seeds used for
transgenic
preparation was from Pusa Ruby variety (PR) of tomato plants. Variety was
developed
by The Indian Agricultural Research Institute (TART), Delhi
[00111] Tomato (Solanum lycopersicum) seeds were used for growing explains
that
were subjected to Agrobacteri urn turnefaciens transformation to study the
nsHb over
expression in the developed transgenic tomato lines. The construct preparation
step
involved the use of set of primers, pEarley Gate 201 expression vector, Trizol
reagent
(Thermo Fisher Scientific), Verso cDNA Synthesis Kit (Thermo Fisher
Scientific),
Phusion0 High-Fidelity DNA Polymerase (M0530). The transformation step
involved
the use of reagents like 100% ethanol, 4% sodium hypochlorite solution, MS
media
(Murashige and Skoog, 1962), pre-incubation media (3% (vv/v) sucrose, 100 M
acetosyringone and 2mg/1 BAP), shoot induction medium (2mg/L zeatin and 250
mg/ml cefotaxime, 3% (w/v) sucrose and 2 mg/L BASTA (plant selection), rooting
media (2mg/L indole acetic acid and 250 mg/ml cefotaxime, 3% (w/v) sucrose and
2
mg/L BASTA), sterile double distilled water and Whatman filter paper for
drying
explants. Electrophoresis, real-time PCR and western blot analysis were
performed for
screening of transgenic tomato plants. 4-Amino-5-Methylamino-2',7'-
Difluorofluorescein diacetate (DAF-FM DA) test was performed to assess NO
levels
inside the plants and parts.
EXAMPLE 1
Construction of nsHb over expressing transgenic tomato plants
[00112] Construct preparation: Solanum lycopersicum non-symbiotic hemoglobin
class 1 (SINSHI) gene having the nucleic acid fragment as set forth in SEQ ID
NO: 1
was amplified by using forward CACCATGAGTAGCTTTAGTGAAGA (SEQ ID
NO: 3) and reverse TAGCACACAAATTAGATTAT (SEQ ID NO: 4) primer. The
PCR product obtained was then cloned behind CaMV2x35S promoter in pEarley Gate
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201 expression vector as shown in the Figure 8 of the present disclosure, to
make the
nsHb over expression construct (recombinant construct as per the present
disclosure).
RNA was isolated by Trizol reagent (Thermo Fisher Scientific) according to
manufacturer's instructions. Total 1 [ig of RNA was used to perform reverse
transcription by using Verso cDNA Synthesis Kit (Thermo Fisher Scientific).
The
cDNA (1 I) obtained was then used to amplify DNA fragments in a 50 vtl
reaction
with PhusionC) High-Fidelity DNA Polymerase (M0530) by using semi-quantitative
PCR. The confirmation of the nsHb over expression construct was done by PCR
(gene
specific primers) and sequencing before the transformation. For more stringent
confirmation, western blot analysis under normal and hypoxic conditions were
performed.
[00113] Tomato transformation: To prepare transgenic plants, tomato
transformation
was achieved by Agrobacterium tutnefaciens infection method. For the purpose
of the
present disclosure, the Agrobacterium tumefaciens (host cell) comprising the
recombinant vector, wherein the recombinant vector comprised the
overexpression
construct (recombinant construct) was used. To carry out tomato
transformation,
tomato seeds were surface sterilized in 100% ethanol for 3 minutes, and then
rinsed
three times with sterile double distilled water, and sequentially placed in 4%
sodium
hypochlorite solution for 5 minutes. The seeds were again rinsed with sterile
double
distilled water and plated on half-strength MS media (Murashige and Skoog,
1962) for
germination. After 10 days, when the cotyledons were completely opened,
seedlings
were suitable to be used as expl ants for the A grobacteriutn tumefaciens
transformation.
Single colony of Agrobacterium tumefaciens (EHA105) carrying nsHb over
expression
construct (recombinant construct) were grown at 28 C with 200 rpm shaking for
48
hours. The cultures were centrifuged at 9,000 g for 10 minutes and the pellet
was re-
suspended in liquid MS medium to 0D600 = 0.6. Cotyledon edges were cut from
both
ends, and the central part of the cotyledons was used as explants for
transformation.
Cotyledons were then incubated for 2 days in pre-incubation media (3% (w/v)
sucrose,
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100 M acetosyringone and 2mg/1 BAP). After pre-incubation, all explanis were
dipped
into Agrobacterium tumefaciens (ETIA105) suspensions and gently shaken for 20
minutes to ensure a full contact between the explants and the bacterial cells.
Explains
were dried on sterilized Whatman filter paper and then transferred to the co-
cultivation
medium followed by incubation for 2 days in the dark at 26 C. Pre-incubation
medium
and co-cultivation medium have the same composition as mentioned in the
materials
and methods of the present disclosure. A.fter 2 days of co-culture with
Agrabacterium
(EH A105), ex pl ants were transferred to the shoot induction medium
containing 2mg/I,
zeatin and 250 nigiral cefota.xime, 3% (w/v) sucrose and 2 ing/l, BASTA (plant
selection). The plates were then incubated at 28 'C with a 16 hours
photoperiod.
Explants were then sub-cultured to fresh media every 12 days. Shoots
regenerated from
shoot induction medium were transferred to rooting media (2mg/L IAA and 250
mg/m1
cefotaxime, 3% (w/v) sucrose and 2 rrig/L BAS'I'A) and after rooting occurs in
the
plants, they were transferred to pots for further growth as shown step wise in
Figure
in, lb and lc.
EXAMPLE 2
Screening of transgenic tomato plants
[00114] In the previous example, the transgenic tomato plants were developed
by using
pEarleyGate 201 vector, which encoded a kanamycin resistance cassette for
plasmid
selection in bacteria along with a bar gene cassette encoding herbicide
resistance. The
bar gene sequence in the T-DNA, was meant for the selection of transformed
plants.
The bar gene from Streptomyces hygroscopicus encodes the enzyme
phosphinothricin
acetyltransferase and confers resistance to herbicides containing
phosphinothricin or
glufosinate. Glufosinate is an inhibitor of glutamine synthetase, which acts
as a central
enzyme for nitrogen metabolism in plants. Based on the factual knowledge about
phosphinothricin being an active ingredient of the commercial herbicide Basta,
the
present disclosure has employed polymerase chain reaction to screen transgenic
tomato
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plants by using BASTA primers specific to the bar gene as the positively
transformed
lines has a bar gene cassette as a part of T-DNA which provided confirmation
for
successful transformation. GoTaq Green Master Mix (M712) was used to conduct
the
PCR reaction according to manufacturer instruction. DNA (100-200 ng) was mixed
with nuclease free water to make up the reaction mixture volume up to 12.5 for
the
loading purpose. 1% Agarose gel was made, and the reaction mixtures were
loaded
onto the gel for electrophoresis. Figure 2a depicts the gel pictures of the
electrophoresis
conducted as a confirmatory test. Real-time PCR and western blot were also
performed
to confirm the developed transgenic lines as shown in Figure 2b and 2c
respectively.
The transgenic lines thus obtained were capable of producing the non-symbiotic
hemoglobin (SEQ ID NO: 2) which can be observed in Figure 2c.
EXAMPLE 3
Resulting features of transgenic tomato plants
[00115] Reduced nitric oxide: Transgenic tomato plants showed reduced nitric
oxide
(NO) levels because of their ability to scavenge NO. NO levels were monitored
using
4-Amino-5-Methylamino-2,7-Difluorofluorescein diacetate (DAF-FM DA) test. DAF-
FM DA test detects and quantifies low concentrations of nitric oxide as when
DAF-
FM reacts with NO, it produces fluorescent benzotriazole that can be detected
by any
fluorescein detector. The results from the DAF-FM DA test carried out in the
present
example, showed that the transgenic tomato plants exhibit reduced levels of NO
as
compared to wild type (WT) tomato plants. Figure 3a shows the NO levels as per
the
DAF-FM fluorescence test results of wild type (WT) and sample transgenic
tomato
plants. The difference between the WT and the transgenic tomato plants was
seen in
relative difference of quantified NO fluorescence intensity in the WT and the
sample
overexpression lines as shown in Figure 3b. Gaseous NO levels were also
measured by
gas-phase chemiluminescence in both the WT and transgenic tomato plants to
study
the NO level differences as shown in Figure 3c. Hypoxic conditions inside the
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developed fruits from the transgenic tomato plants were confirmed from the
observations recorded for a decrease in internal oxygen with time as shown in
Figure
4.
[00116] Increased fruit number, reduced fruit size and weight: The observed
phenotype
of fruits developed from transgenic tomato plants (transgenic plant as per the
present
disclosure) showed a decrease in fruit size. The number of fruits increased
per plant in
transgenic tomato plants as shown in Figure 5 and Table 1. It can be observed
from
Table 1 that the transgenic plant ¨ nsfib over expressing tomato transgenic
line 7 (0E7)
shows the number of tomato fruits which is at least 2-3 folds as compared to
the non-
transgenic or WT tomato plant.
[00117] It can be observed from Table 2 that the average weight of tomato
produced
by the nsHb over expressing tomato transgenic plant lines 0E7 and 0E5 is more
than
two folds lesser than the wild type plants, therefore, providing fruits that
were lighter
in size as compared to that of the wild types.
[00118] The nsHb over expressing transgenic tomato plants (0E7 and 0E5) also
showed reduction in diameter of fruits giving cherry like appearance as shown
in Figure
5. Table 3 data shows the extent of difference between the diameter of tomato
fruits
produced from transgenic tomato plants and WT plants. It can be observed that
in some
cases (Table 3), that the extent of reduction in diameter of the tomato fruits
is as high
as 9 folds in the transgenic plants as compared to that of the wild type.
[00119] Table 1. Number of fruits per plant in wild type plants and nsHb over
expressing transgenic tomato plant lines.
WT nsHb OE 7
6 30
11 18
7 28
3 23
2 25
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8 17
9 23
9 25
[00120] Table 2. Fruit weight (gm) of fruits in wild type plants and nsHb over
expressing transgenie tomato plant lines.
WT nsHb OE 7 nsHb OE 5
49.16 6.65 7.069
31.3 11.05 9.33
54.62 10.25 11.07
29.74 10.28 12.44
47.6 26.16 8.82
23.39 10.21 6.05
42.38 7.29 10.22
19.71 11.58 7.66
[00121] Table 3. Fruit diameter in in wild type plants and ns lh over
expressing
transgenic tomato plant lines.
S.No. WT nsHb OE 7 nsHb OE 5
1 8.96 0.66 1.13
2 4.44 1.86 1.44
3 10.83 1.72 1.43
4 3.81 L83 1_64
5 5.47 3.22 0.73
6 2.5 1.18 0.92
7 5.1 0.64 1.29
8 2.04 1.79 0.77
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[00122] Early fruit ripening: In the present disclosure, the developed
transgenic tomato
plants showed early ripening of fruits which may be due to lesser nitric oxide
(NO)
levels in the transgenic plants overexpressing non-symbiotic hemoglobin gene.
It
suggests that the NO was involved in the ripening of tomato fruits which
inhibits
ethylene production. Therefore, reduced NO levels in the fruits from the
developed
transgenic tomato plants triggered the early ripening phenomenon. Figure 6b
and
Figure 6d of the present disclosure showed images for the early ripening of
tomato
fruits from the OE lines 7 & 5 as disclosed herein. For a better comparison to
highlight
the early ripening and higher yield of tomato fruits in nsHB overexpressing
lines,
images of fruits developed from the wild type tomato lines were shown in
Figure 6a
and 6c respectively. The fruits obtained from developed transgenic tomato
plants as
shown in Figures 6b and 6d, were matured 3-4 weeks before than wild type (WT).
[00123] To establish the fact of obtaining early ripened tomatoes from the
developed
transgenic lines as per the present disclosure, a virtual test was conducted
for six
consecutive days to record the relative ripening progress in the wild type
(WT) and
developed transgenic tomato plants (transgenic plants as per the present
disclosure),
the transgenic produce showed early ripening in the test against the WT as in
over
expression line 7, the prominent start of ripening was visible from the fourth
day itself,
while in the WT, the fruit was not even partially ripened on the sixth day,
however the
fruits from over expression line 7 were fully ripened at the end of sixth day
as shown
in Figure 7.
EXAMPLE 4
GC-MS analysis of tomato fruits
[00124] Lyophilized leaf and fruits samples of both wild type (WT) and
developed
transgenic tomato plants of the present disclosure, were weighed and extracted
according to a method as described in Kundu, A., Mishra, S., & Vadassery, J.
(2018).
Spodoptera litura-mediated chemical defense is differentially modulated in
older and
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younger systemic leaves of Solanum lycopersicum. Planta, 248(4), 981-997, with
a
slight modification. In brief, 20 mg lyophilized leaf/ fruits were extracted
in 480 I of
GC grade methanol (MERCK, USA), and 20 1 of 0.2 mg/ml ribitol (adonitol) was
added to it as an internal standard, to obtain a mixture. Mixture produced was
vigorously shaken fur 2-3 min and then incubated at 70 "C for 15 min.
Thereafter, 500
I of water was added and vigorously shaken for few times, followed by the
addition
of 250 1 of chloroform (MERCK, USA) and mixed thoroughly. The mixture was
centrifuged at 2200g for 10 min at room temperature (¨ 28 C). Thereafter, the
upper
aqueous phase was carefully taken out and dried in speed vacuum at 45 C.
Dried
fraction was then re-dissolved by vortexing in 40 1 of 20 mg/ml methoxamine
hydrochloride in pyridine and then incubated at 37 C for 90 min. Thereafter,
60 I of
MSTFA (N-methyl-N-(trimethylsily1) trifluoroacetamide) was added to the
mixture
and incubated at 37 'V for 30 min. Following derivatization, 2 1 of the
sample was
used to perform gas chromatography¨mass spectrometry (GC¨MS) analysis
employing
a Shimadzu GC¨MS-QP2010Tm coupled with an auto sampler¨auto injector (AOC-
20si). Analysis was conducted by exploiting Rtx-50 capillary column (Restek
Corporation, USA) and helium was used as a carrier gas. The method consisted
of 80
'V isothermal heating for 2 min, followed by ramp rate of 5 C min' to 250 C,
a
withhold of 2 min and a final ramp of 10 C min', a withhold time of 24 min.
Chromatogram integration and mass spectra analysis was done by using GC¨MS
solution software (Shimadzu0) and NIST14s. WILEY8 spectral library was used to
identify deri vati zed m etabo 1 i tes, wherein the derivatives of glucose,
sucrose, and
fructose were confirmed for identification with authentic standards analyzed
in GC¨
MS. Metabolites that were derivatized by different numbers of trimethylsilyl
(TMS)
were considered as a different metabolite. For the metabolite having multiple
peaks,
summation of the peak area was done after confirming spectral data according
to the
Lisec J, Schauer N, Kopka J, Willmitzer L, Fernie AR (2006) Gas chromatography
mass spectrometry-based metabolite profiling in plants. Nat Protoc 1:1-10.
Peak area
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of each of the compounds was normalized by dividing by peak area of internal
standard
(ribitol) in each run.
[00125] RESULTS
[00126] Untargeted metabolite analysis was performed from the fully ripened
fruits of
WT and nsHb OE plank (Figure 9), wherein five replicates per plant were taken
from
four plants (both WT and transgenic tomato plants). Referring to Figure 9, it
can be
observed that most of the metabolites from WT and nsHb OE fruits did not show
a
significant difference, however, the major source of variation was found in
the levels
of sugars. Figure 10 shows a comparison in the levels of sugars found in the
fruits of
WT and transgenic tomato plants. Referring to Figures 10a to Figure 10d, it
can be
observed that sugars such as Sucrose, D-Allose, Mannobiose, D-Arabinose, D-
glucofuranoside were highly accumulated in the fruits of nsHB overexpressing
(OE)
plants (transgenic tomato plant) in comparison to the fruits of WT plants. It
was also
observed that there was a higher accumulation of myo-inositol (1,2,3,4,5,6
Hexa-0
Trimethelsilyl Myo-Insositol) fruits of nsHB overexpressing (OE) plants
(transgenic
tomato plant) in comparison to the fruits of WT plants. Such a higher
accumulation of
myo-inositol can be helpful to regulate Insulin levels, and hence the tomato
fruit of the
transgenic plant can be useful as food for people with Type-2 Diabetes.
Similarly,
Ribofuranose and Galactopyranose showed higher accumulation in nsHb OE fruits
in
comparison to WT fruits. Hence, the fruit of the transgenic tomato plant of
the present
can be deployed in a food product.
EXAMPLE 5
Inductively coupled plasma mass spectrometry (ICP-MS)
[00127] To perform ICP-MS (Agilent 7800), lyophilized powdered samples of
tomato
fruits (from both WT and transgenic plant of the present disclosure) were
subjected to
microwave-assisted acid digestion. Briefly, 0.2 g of each lyophilized powdered
sample
was accurately weighed and inserted directly into microwave digestion vessels,
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followed by the addition of 8.0 ml of concentrated nitric acid (70%). The
digestion
procedure was performed under the following parameters: Power used was 1200 W,
ramp time was 20 min, hold time was of 20min and temperature of the oven was
180
'C. After cooling, the contents of the tubes were transferred to 50 ml self-
standing
polypropylene volumetric tubes (Tarsons, India). The contents were made up to
50 iril
with water (Malique). Subsequently, the samples were filtered with 0.2311
syringe filter
and diluted in the ratio of 1:10 with 2% HNO3. For phosphate analysis, samples
were
diluted at the ratio of 1:100. Processed samples were then analysed.
[00128] RESULTS
[00129] To investigate the effect of over-expression of nsHbl gene (SEQ ID NO:
1)
upon nutritional value of tomato fruits, ICPMS analysis of fully ripened
fruits of WT
and nsHbl over-expression plants (transgenic tomato plants of the present
disclosure)
was performed. Results of the ICPMS analysis as depicted in Figure 11 show
that
elements like Sodium (Na), Magnesium (Mg), Potassium (K) and calcium (Ca) were
higher in nsHbl over-expression fruits as compared to WT fruits, however, no
significant difference was found in the concentrations of Nickle (Ni), Couper
(Cu) and
Zinck (Zn). Contrary to the higher concentration of some of the elements as
mentioned
above, a reduced concentration of Arsenic (As) was found in nsHbl over-
expression
fruits in comparison to WT tomato fruits. This indicates that the expression
of nsHbl
gene (SEQ IN NO: 1) in the fruits from the transgenic tomato plant leads to
enhanced
nutritional value by enhancement of Myo-Inositol, Na, Mg, K and Ca useful for
consumption.
[00130] Therefore, it can be concluded that the transgenic tomato plants over-
expressing nsHbl gene (SEQ ID NO: 1) leads to: (i) early ripening of tomato
fruits;
(ii) reduction in NO levels, (iii) increase in fruit number: (iv) reduced
size; (v) reduced
weight; (vi) higher accumulation of sugars, such as derivatives of glucose,
sucrose, and
fructose; and (vii) tomato fruits with better nutritional properties, as
compared to WT
tomato fruits. Due to the enhanced nutritional properties, the fruit from the
transgenic
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plant is suitable for consumption in the form of a food product, suitable for
human
consumption.
[00131] Although, the examples are shown with tomato, however, it can be
contemplated that overexpression of non-symbiotic hemoglobin gene in
climacteric
fruit-bearing plants could show similar phenotypic effects as shown by tomato.
For
example, overexpression of non-symbiotic hemoglobin gene in banana would yield
in
a transgenic plant having early ripening banana fruits. Similar methods can be
followed
for other plants like mango, sugar apple, peach, and apricot.
Advantages of the present disclosure
[00132] The above-mentioned implementation examples as described on this
subject
matter and its equivalent thereof have many advantages, including those which
are
described.
[00133] The developed transgenic tomato plants (transgenic plants as per the
present
disclosure) shows increased number of fruits per plant, which can be directly
correlated
with the enhanced yield per plant. Enhanced yield per plant will clearly help
the farmers
and the consumer sector as well. Higher yield can also solve the problem to
balance a
ratio between the fruit production and consumption to match the present-day
needs.
Early ripening achieved in the developed transgenic lines will help farmers to
grow
multiple crops every year due to the shorter life span of the developed lines.
In fact,
early ripening of fruits will also minimize the use of harmful artificial
ripening agents
which poses deteriorating impacts on the human health. Cherry-like appearance
of the
transgenic fruits as per the present disclosure will help to satisfy the needs
of various
consumer segments in particular, as it can be specifically used in salads or
pizzas, etc.
Since it is a well-known fact that tomatoes with cherry-like appearance are
preferred
for culinary decoration, therefore, the transgenic tomato lines as per the
present
disclosure would satisfy the requirements of cherry tomatoes in hotels, and
restaurants.
Since cherry tomatoes are difficult to produce and much harder to maintain,
the
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transgenic tomato plant as per the present disclosure has a significant
advantage in
terms of the uniform production of cherry-sized tomato fruits and can be an
economically viable option as compared to cherry tomatoes. The reduced size
and fruit
weight will also help in the effective transportation of the final product.
High yield will
be very impactful in minimizing the losses incurred through spoilage of the
fruits
during transportation, high yield will compensate for the damages occurred.
The small
size of the tomatoes will also help to reduce the spoilage through fruit
cracks as
generally seen in wild tomatoes.
15
25
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