The present invention relates to an insect-free method of producing enation leaf curl
disease in okra plants using inoculation with agrobacterium containing cloned DNAs
5 of the virus and satellite responsible for the disease.
BACKGROUND INFORMATION AND PRIOR ART
The term ‘agroinoculation’ implies the use of Agrobacterium tumefaciens or A.
10 rhizogenes for transferring cloned viral DNA in plants to produce infection with the
virus. In plants, apart from whitefly transmission, methods such as mechanical
inoculation and particle bombardment provide alternative ways of producing virus
infections from cloned viral DNA. However, agroinoculation is one of the most
effective ways of obtaining viral infections. It involves transferring viral DNA cloned
15 in binary plasmid into the host plant. These ‘agroinfectious’ clones are powerful tools
to study the viral host range, trans-replication of viral DNA components and genetic
determinants of symptom development. Agroinoculation has emerged as a popular
tool for performing mutational studies with the viral genome, to study its local and
systemic movement and the mapping of viral genes involved in replication. Such
20 experiments are a great help in testing the efficacy of various viral resistance
strategies under laboratory conditions.
Geminiviruses are single stranded DNA viruses, having small genomes, which infect
plants in the tropical and subtropical regions of the world. Agroinoculation has been
shown to efficiently transmit many geminiviruses such as bhendi yellow vein mosaic
25 virus (Jose and Usha, 2003), mungbean yellow mosaic virus (Jacob et al., 2003;
Usharani et al., 2005), tomato golden mosaic virus (Hayes et al., 1988), tomato
yellow leaf curl virus (Navot et al., 1991), Sri Lankan cassava mosaic virus (Mittal et
al., 2008), chilli leaf curl virus (Chattopadhyay et al., 2008) and vernonia yellow vein
3
virus (Packialakshmi and Usha, 2011) to their natural hosts. Geminiviruses are
responsible for some of the most important viral diseases of plants, such as the
Enation leaf curl disease (ELCuD) of okra (Abelmoschus esculentus, family
Malvaceae), a disease which is currently threatening okra production in large parts of
5 India. The geminivirus Okra enation leaf curl virus (OELCuV) has been reported to
be associated with ELCuD-affected okra plants and is believed to be responsible for
causing the disease (Naresh et al., 2019). Since okra is a popular vegetable consumed
in India and the neighboring countries, it is important to increase okra production by
developing varieties and hybrids which have resistance against ELCuD, a process
10 which requires screening of a large number of okra plants for resistance. This patent
describes a simplified method and provides the materials for the above screening
process.
The traditional method for screening of resistance against ELCuD in okra involves
inoculation with whiteflies, a process which is seasonal and time-consuming. The
15 present invention relates to the use of the method of agroinoculation as a means for
quick screening procedure for genetic resistance in okra plants against ELCuD.
A patent search revealed the following two patents, with some similarity to the
present invention. However, each of the patent mentioned is substantially different
from the present invention. 20 CN110358790 discloses a method for quick genetic transformation of virus infection
of plants. This patent describes a method of inoculation of tomato plants with a
cloned viral DNA, which is different from OELCuV. The method described is also
very different from the one described in the present invention.
US20060037105 discloses a root agroinoculation method for virus induced gene
25 silencing. This patent describes a method of agrobacterium-mediated inoculation of
laboratory tobacco (Nicotiana benthamiana) plants with a virus-derived DNA. Both
the viral DNA and the method described are very different from the present
invention.
4
References
Chattopadhyay, B., Singh, A. K., Yadav, T., Fauquet, C. M., Sarin, N. B. &
Chakraborty, S. (2008). Infectivity of the cloned components of a begomovirus: DNA
beta complex causing chilli leaf curl disease in India. Arch Virol 153, 533–539.
5 Ghosh, R., Paul, S., Ghosh, S.K. & Roy A. (2009). An improved method of DNA
isolation suitable for PCR-based detection of begomoviruses from jute and other
mucilaginous plants. J Virol Methods 159, 34-39.
Hayes, R. J., Coutts, R. H. A. & Buck, K. W. (1988). Agroinfection of Nicotiana spp.
10 with cloned DNA of tomato golden mosaic virus. J Gen Plant Pathol 69.
Jacob, S. S., Vanitharani, R., Karthikeyan, A. S., Chinchore, Y., Thillaichidambaram,
P., & Veluthambi, K. (2003). Mungbean yellow mosaic virus-Vi agroinfection by
codelivery of DNA A and DNA B from one Agrobacterium strain. Plant Dis 87, 247- 15 251.
Jose, J. & Usha, R. (2003). Bhendi yellow vein mosaic disease in India is caused by
association of a DNA β satellite with a begomovirus. Virology 305, 310–317.
Mittal, D., Borah, B. K. & Dasgupta, I. (2008). Agroinfection of cloned Sri Lankan
cassava mosaic virus DNA to Arabidopsis thaliana, Nicotiana tabacum and cassava.
20 Arch Virol 153, 2149–2155.
Naresh, M., Khan, Z. A., Kumar, R., Kale, S. P., Patil, V. M., Rajput, J. C. and
Dasgupta, I. (2019) Occurrence and variability of begomoviruses associated with
bhendi yellow vein mosaic and enation leaf curl diseases in south-western India.
VirusDisease 30, 511-525.
25 Navot, N., Pichersky, E., Zeidan, M., Zamir, D. & Czosnek, H. (1991). Tomato
yellow leaf curl virus: A whitefly-transmitted geminivirus with a single genomic
component. Virology 185, 151–161.
Packialakshmi, R & Usha, R. (2011). A simple and efficient method for agroinfection
of Vernonia cinerea with infectious clones of Vernonia yellow vein virus. Virus
30 genes 43, 465-470.
Usharani, K., Surendranath, B., Haq, Q. & Malathi, V. G. (2005). Infectivity analysis
of soybean isolate of mungbean yellow mosaic India virus by agroinoculation. J Gen
Plant Pathol 71, 230-237.
35
5
OBJECTS OF THE PRESENT INVENTION
It is therefore an object of the present invention to provide a method of
agroinoculation of okra plants using cloned DNA of OELCuV and BYVMB.
5
Another object of this invention is to provide the sequences of DNA of OELCuV to
construct the agroinfectious clone (SEQ ID NO. 1).
Another object of this invention is to provide the sequences of cloned partial dimer of
OELCuV which is called as agroinfectious clone (SEQ ID NO. 2).
10 Another object of this invention is to provide sequences of the abutting primers based
on SEQ ID NO. 1 used for the construction of agroinfectious clones (SEQ ID NO. 3
and SEQ ID NO. 4).
Yet another object of this invention is to provide gene sequences of the primers for
amplification of the coat-protein gene of OELCuV by PCR (SEQ ID NO. 5 and SEQ
15 ID NO. 6).
Yet another object of this invention is to provide gene sequences of the abutting
primers for full length viral DNA amplification of OELCuV by PCR (SEQ ID NO. 7
and SEQ ID NO. 8).
Still another object of this invention is to provide gene sequences of the primers for
20 amplification of OELCuV C4 gene by PCR (SEQ ID NO. 9 and SEQ ID NO. 10).
Still another object of this invention is to provide gene sequences of the primers for
amplification of BYVMB by PCR (SEQ ID NO. 11 and SEQ ID NO. 12).
Further object of this invention is to provide the sequences of DNA of BYVMB to
construct the agroinfectious clone of BYVMB (SEQ ID NO. 13).
6
Further object of this invention is to provide the sequences of cloned partial dimer of
BYVMB (SEQ ID NO. 14).
Yet further object of this invention is to provide sequences of the abutting primers
based on SEQ ID NO. 13 used for the construction of agroinfectious clones (SEQ ID
5 NO. 15 and SEQ ID NO. 16).
Still further object of this invention is to provide a method for agroinoculation into
okra plants for the purpose of studying infectivity of the cloned OELCuV and
BYVMB DNAs.
The final object of this invention is to provide a method of assay for the accumulation
10 of the DNAs of OELCuV and BYVMB, whose infectivity is being studied, using
PCR and DOT-BLOT analysis.
SUMMARY OF INVENTION
The present invention relates to the development of an agroinoculation method for
15 okra to obtain plants infected with OELCuV and BYVMB.
The present invention provides information about the construction of partial dimer
agroinfectious clones using DNA of OELCuV. The invention further provides an agroinfectious clone, in which partial dimer of
BYVMB is cloned.
20 The present invention provides a method for agroinoculation of okra plants with the
OELCuV and BYVMB agroinfectious clones.
7
The present invention also provides information about the assay to detect
accumulation of the DNAs of OELCuV and BYVMB in agroinoculated plants using
dot-blot analysis.
The invention further relates to the use of this technique to study infectivity of
5 geminiviruses in okra plants and molecular analysis of viral DNA accumulation
levels by using PCR and Dot-Blot analysis.
Thus, the present invention describes a method and provides the cloned DNAs for a
process of infecting okra plants, using agrobacterium, with OELCuV and BYVMB to
10 cause ELCuD. The present invention, thus, provides a means of screening large
numbers of okra plants against ELCuD, which is a mandatory step in breeding for
resistance against ELCuD. The invention comprises three parts; in part one, the
construction of infectious clones of OELCuV and MYVMB are described, in which
the viral and satellite DNAs are cloned in a special manner in a binary plasmid; in
15 part two, the method of inoculation of the constructs to okra plants through
agrobacterium is described and in part three, the process of confirming infection of
okra plants with OELCuV and BYVMB is described, using PCR amplification and
DNA dot-blot. Hence, this invention makes it possible for any user to inoculate large
number of okra plants and to determine their resistance levels against ELCuD in a
20 matter of few weeks. This invention makes testing of okra plants possible year-round
and independent of using whiteflies, which, otherwise is necessary for testing of
ELCuD resistance.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
25
The illustrated embodiments of the subject matter will be best understood by
reference to the drawings, wherein like parts are designated by like numerals
throughout. The following description is intended only by way of example, and
8
simply illustrates certain selected embodiments of devices, systems, and processes
that are consistent with the subject matter as claimed herein, wherein:
FIG 1 shows a diagrammatic representation of agroinfectious clones of OELCuV and
5 BYVMB: (A) OELCuV1.5mer and (B) BYVMB1.1mer. Arrows indicate the
orientation of the genes. The names of each gene (block arrows) are mentioned.
Rectangles indicate functionally important regions (IR: intergenic region, A-Rich:
region rich in A residues, SCR: satellite conserved region). Symbols above IR and
SCR indicate stem-loop region. The positions of some relevant restriction
10 endonuclease cleavage sites and their coordinates are indicated.
FIG 2 shows okra plants showing symptoms of (A) Upward leaf curling, (B) bud
clumping, (C) vein thickening (D) profuse branching and (E) petiole bending and
stunting at 22 days post inoculation (dpi) with OELCuV1.5mer and BYVMB1.1mer. 15 In panel C, leaf shown in the left was from a mock-inoculated plant. In panel E, the
plant on the right was mock-inoculated and shows no symptoms, whereas the plant on
the left and the centre were inoculated with OELCuV1.5mer and BYVMB1.1mer.
FIG 3 shows the genetic maps of OELCuV and BYVMB and the positions of primers
20 (arrows) used for PCR-based amplification. Panel A) primers for coat protein gene of
OELCuV & C4 gene, panel B) primers for βC1 gene of BYVMB. Panel C) explains
how abutting primers from OELCuV are used to determine release of full-length viral
DNA upon inoculation to okra plants. The map in the right represents the
OELCuV1.5mer, the position is indicated by the double-headed arrow, which is
25 shown in detail in the top left. The lower left portion shows the expected sizes of
amplified DNA when the viral DNA is released (2.7 kb) or not released (12.5 kb).
9
FIG 4 shows the electrophoresed gel photographs of PCR products conducted on okra
plants agroinoculated with various constructs using four sets of primers described in
FIG 3. Panel A shows the PCR products using CP primers; gels i, ii and iii represent
plants inoculated with OELCuV1.5mer and BYVMB1.1mer; gel iv represent plants
5 inoculated with OELCuV1.5mer only and gel v represents plants inoculated with
OELCuV1.5mer and BYVMB1.1mer with E. coli (mock-inoculation). In gel iv, two
lanes were loaded with samples from naturally-infected okra plants, as controls (NI).
Panel B shows PCR products using C4 primers; gels i and ii represent plants
inoculated with OELCuV1.5mer and BYVMB1.1mer, whereas gel iii represents
10 plants mock-inoculated, as in panel A v. Panel C shows the PCR products using βC1
primers in both the gels representing plants inoculated with OELCuV1.5mer and
BYVMB1.1mer (lane E.c. represents mock-inoculated sample). Panel D shows PCR
products using abutting primers, the inoculation history of samples loaded in lanes
being shown on top of each gel [M: Marker/DNA Ladder, +: positive control DNA, - 15 : negative control DNA, NI: Naturally infected DNA, E.c.: DNA from E. coli
inoculated plant sample].
FIG 5 shows the results of DNA dot-blot analysis of okra plants inoculated with
OELCuV1.5mer and BYVMB1.1mer; Row 1: Cloned OELCuV plasmid DNA (25
20 nanograms), Row 2: Mock inoculated plant DNA (1 microgram); Row 3-5: DNA
from plants inoculated with OELCuV1.5mer and BYVMB1.1mer. The probe for
hybridization was derived from 770 bp coat protein gene fragment of OELCuV. Each
sample was spotted in duplicate (represented in the columns at the left and right).
25 Table 1 shows a summary of the results of analysis of agroinoculated okra plants
using the set of cloned DNAs as indicated.
Table 2 shows the short sequences and the primers applicable.
10
DETAILED DESCRIPTION OF THE INVENTION
At the very outset of the detailed description, it may be understood that the ensuing
description only illustrates a form of this invention. However, such a form is only
5 exemplary embodiment, and without intending to imply any limitation on the scope
of this invention. Accordingly, the description is to be understood as an exemplary
embodiment and teaching of invention and not intended to be taken restrictively.
Throughout the description and claims of this specification, the phrases “comprise”
10 and “contain” and variations of them mean “including but not limited to”, and are not
intended to exclude other moieties, additives, components, integers or steps. Thus, the
singular encompasses the plural unless the context otherwise requires. Wherever
there is an indefinite article used, the specification is to be understood as
contemplating plurality as well as singularity, unless the context requires otherwise.
15
Thus, the terms “comprises”, “comprising”, or any other variations thereof used in
the disclosure, are intended to cover a non-exclusive inclusion, such that a device,
system, assembly that comprises a list of components does not include- only those
components but may include other components not expressly listed or inherent to
20 such system, or assembly, or device.
In other words, one or more elements in a system or device proceeded by
“comprises… a” does not, without more constraints, preclude the existence of other
elements or additional elements in the system, apparatus or device.
25
Features, integers, characteristics, compounds, chemical moieties or groups described
in conjunction with an aspect, embodiment or example of the invention are to be
understood to be applicable to any other aspect, embodiment or example described
11
herein unless incompatible therewith. All the features disclosed in this specification
(including any accompanying claims, abstract and drawings), and/or all of the steps
of any method or process so disclosed, may be combined in any combination, except
combinations where at least some of such features and/or steps are mutually
5 exclusive. The invention is not restricted to the details of any foregoing embodiments.
The invention extends to any novel one, or any novel combination, of the features
disclosed in this specification including any accompanying claims, abstract and
drawings or any parts thereof, or to any novel one, or any novel combination, of the
steps of any method or process so disclosed.
10
The reader's attention is directed to all papers and documents which are filed
concurrently with or before this specification in connection with this application and
which are open to public inspection with this specification, and the contents of all
such papers and documents are incorporated herein by reference. Post filing patents,
15 original peer reviewed research paper shall be published.
The following descriptions of embodiments and examples are offered by way of
illustration and not by way of limitation.
20 Unless contraindicated or noted otherwise, throughout this specification, the terms
“a” and “an” mean one or more, and the term “or” means and/or. As used in the
description herein and throughout the claims that follow, the meaning of “a.” “an,”
and “the” includes plural reference unless the context clearly dictates otherwise. Also,
as used in the description herein, the meaning of “in” includes “in” and “on” unless
25 the context clearly dictates otherwise.
12
A preferred embodiment of the present invention relates to the development of an
agroinoculation method for okra and its use, derived from cloned OELCuV and
BYVMB.
5 Another embodiment of the present invention relates to providing the nucleotide
sequences of OELCuV for the construction of agroinfectious clone used for the
development of agroinoculation method (SEQ ID NO. 2).
Yet another embodiment of the present invention relates to providing the nucleotide
10 sequences of BYVMB for the construction of agroinfectious clone used for the
development of agroinoculation method (SEQ ID NO. 14).
Yet another embodiment of the present invention relates to the process of cloning
partial dimers of OELCuV and BYVMB.
15
Yet another embodiment of the present invention relates to the transformation of the
partial dimers of OELCuV and BYVMB into agrobacterium cells.
Yet another embodiment of the present invention relates to the method of
20 agroinoculation of plants, where the plant can belong to the family Malvaceae.
Yet another embodiment of the present invention relates to the process of inoculation
of agrobacterium cells containing the DNA constructs of OELCuV and BYVMB into
the region of growing shoots in okra plants.
25
Yet another embodiment of the present invention relates to the composition of
agroinfiltration medium used for agroinoculation of okra plants.
13
Yet another embodiment of the present invention relates to the providing the
nucleotide sequences of the primers used for molecular analysis of viral DNA
accumulation levels from okra plants (SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,
SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10).
5
Yet another embodiment of the present invention relates to providing a method for
agroinoculation into okra plants for the purpose of producing infection with the
cloned viral and satellite DNAs.
10 Finally, another embodiment of the present invention relates to providing a method of
inoculation of the cloned partial dimers of OELCuV and BYVMB which are
infectious, using PCR and DNA dot-blot analysis.
The construction of agroinfectious clones of OELCuV and BYVMB were performed
15 as described in Example 1 and confirmed by plasmid isolation and restriction
digestion with several enzymes. Okra plants, which were 30 days old, were
agroinoculated with a combination of OELCuV and BYVMB. This was performed by
injecting 100 μl of a suspension of agrobacterium cells containing the cloned DNAs
of OELCuV and BYVMB in the shoot apical meristem region of the plant. The
20 process of agroinoculation is described in Example 2. For negative control, mockinoculations were performed, wherein, instead of agrobacterium cells, E. coli cells,
carrying the same plasmid constructs were used for inoculation. After 18-20 days’ post inoculation (dpi), about 60% plants started to show symptoms
25 of leaf curling, petiole bending, severe lateral branching and clumping, bending of
stem, and severe thickening of veins as shown in FIG 2.
14
To determine the viral DNA accumulation, PCR analysis was performed as described
in Example 4 using 4 sets of primers as shown schematically in FIG 3. Total DNA
was isolated at 22 dpi from newly-emerged leaves of agroinoculated okra plants as
described in Example 3. All four sets of primers were designed from SEQ ID 1, SEQ
5 ID 2 and SEQ ID 13, the nucleotide sequence of OELCuV, BYVMB and partial
dimer clone of OELCuV respectively. It was seen that more than 62% of all okra
plants tested showed amplification of the DNA of different sizes as shown in FIG 4
indicating presence of the viral DNA molecules in a majority of the inoculated plants. Having ascertained that OELCuV and BYVMB were capable of replicating and
10 accumulating in agroinoculated okra plants, DNA dot–blot analysis was performed.
FIG 5 shows the accumulation of viral DNA in OELCuV and BYVMB co-inoculated
okra plants at 22 dpi using coat protein gene-specific probe. Mock-inoculated sample
did not show any signals.
15 The examples given are merely illustrative of the uses, processes and products
claimed in this invention, and the practice of the invention itself is not restricted to or
by the examples described.
Example 1: Construction of agroinfectious clone of OELCuV and BYVMB.
20
The full-length OELCuV (NCBI Accession number: KJ462074) and the associated
betasatellite (NCBI Accession number: KJ462076) were amplified with a set of
abutting primers (SEQ ID NO. 3 and SEQ ID NO. 4) designed around the restriction
site SacI and cloned in pTZ57R/T (InsTAclone PCR cloning kit, Thermo Fisher
25 Scientific, USA). The clones were confirmed by sequencing (DNA sequencing
facility at Department of Biochemistry, University of Delhi South Campus, New
Delhi) and were named as OELCuV-TA and BYVMB-TA respectively. Both the
clones were further subcloned as partial dimers converted in a manner that the repeat
15
fragment and the full-length fragment are in the same orientation. Also, both clones
were constructed in a way so that the partial dimer had two conserved hairpin
sequences, a defining feature of all geminiviral DNAs indicating the origin of
replication, which facilitates the replicational release of the viral DNA. Both the
5 binary vector clones were mobilized into agrobacterium strain EHA105 and was used
to check the infectivity using the method described in Example 2.
The strategy for construction of OELCuV agroinfectious clone was as follows: The
restriction enzymes SacI–HindIII were used to obtain fragments of approx. 1.2 kb and
1.5 kb from OELCuV-TA clone. The fragments were ligated in binary vector
10 pCambia2300 (SacI-HindIII digested backbone). The clones, thus obtained, were
named as 1.5-bitmer and 1.2-bitmer. The 1.5-bitmer was used for further cloning. For
constructing a partial dimer, the full-length DNA fragment from OELCuV-TA clone
was taken out by restriction digestion with SacI and ligated in 1.5-bitmer in SacI site
and transformed into E. coli DH5α. The obtained colonies were screened by
15 restriction digestion and a clone showing the expected insert was named as
OELCuV1.5mer.
The strategy for construction of BYVMB agroinfectious clone was as follows: The
1.1 kb fragment obtained after SacI–BglII digestion of BYVMB-TA clone was
ligated in binary vector pCambia2300 digested with SacI-BamHI (BglII site is
20 compatible with BamHI site). The ligated product was transformed in E. coli DH5α.
This partial clone was named 1.1-bitmer. Next, the full-length betasatellite fragment
from BYVMB-TA clone was taken out by restriction digestion with SacI, ligated in
SacI digested 1.1-bitmer and transformed in E. coli DH5α. A clone showing the
expected insert in the correct orientation was named as BYVMB1.1mer.
25
Example 2: Growing okra plants and method of agroinoculation.
16
Okra plants were grown in pots containing soil with appropriate natural fertilizers in a
greenhouse, which was maintained between temperatures of 28C and 32C under
natural daylight conditions and supplemented light to give a 16 hours light and 8
hours dark period. The plants at 7-8 leaves stage with visible apical buds were used
5 for inoculation. A colony of agrobacterium containing OELCuV1.5mer and another
containing BYVMB1.1mer were inoculated in 5 ml Luria Bertani (LB) medium
containing 50 μg/ml rifampicin and 50 μg/ml kanamycin. This was incubated in a
shaker for 36 hrs at 28°C in the dark at 200 rpm. After 36 hr, a 250 ml secondary
culture (~0.1% of primary culture as inoculum) was raised in LB medium containing
10 50 μg/ml rifampicin and 50 μg/ml kanamycin and 0.2 M acetosyringone. The
secondary culture was grown overnight at 28°C to attain an O.D.600 of 1.2. The
overnight grown secondary culture was collected by centrifugation at 4307xg for 10
min at room temperature in Sorvall centrifuge LYNX6000 (Thermo Fisher
Scientific). To prepare agroinfiltration medium, the pellet was bacterial resuspended
15 in 1 ml of resuspension buffer containing 10 mM MES [2-(N-morpholino)
ethanesulfonic acid], 10 mM magnesium chloride and 250 μM acetosyringone. The
bacterial suspension was incubated in the resuspension buffer at room temperature for
3 hr. Since both constructs were co-inoculated, it was made sure that each bacterial
culture in the final suspension was collected from a 250 ml secondary culture and
20 resuspended in 1 ml of resuspension buffer. The agroinoculation was performed in
the meristematic tissues of the plant. The terminal buds of the plant were first
punctured using a syringe and 100 μl of re-suspended agrobacterium cells were
injected at the shoot apical meristem region. Following inoculation, the plants were
maintained under the same growing condition till further analysis.
25
Example 3: Isolation of total genomic DNA from newly emerged leaves of
inoculated plants.
17
The total genomic DNA was isolated from newly emerged leaves of agroinoculated
plants by employing a method derived from CTAB-Mucilage-free method (Ghosh et
al., 2009). Leaves were harvested and frozen in liquid nitrogen. One hundred mg of
frozen leaf tissue was ground to a fine powder without liquid nitrogen and 1 ml of
5 pre-warmed (65°C) extraction buffer (100 mM Tris–HCl pH 8, 10 mM EDTA pH 8,
1.4 M NaCl, 2% CTAB, 0.2% β-mercaptoethanol and 2% PVP) was added. The
samples were then transferred into 2 ml microcentrifuge tubes and RNase (20 mg/ml)
was added followed by incubation at 65°C for 45-60 minutes. The contents were
mixed three to four times by inverting the tubes gently. Next, 0.6 volume of
10 chloroform: iso-amylalcohol (24:1) was added for removing the organic contaminants
and mixed to form an emulsion. The aqueous phase was collected by centrifuging the
samples at 16,500xg for 10 min at room temperature (RT). This step was repeated
with 0.5 volume of chloroform: iso-amylalcohol (24:1). Next, about two-thirds of the
volume of chilled iso-propanol was added and mixed well by inverting the tubes. To
15 this 50 μl of ice cold 3M ammonium acetate was added and mixed very slowly for a
couple of times. The samples were incubated at -20°C for 10 minutes. After
centrifugation at 14,000xg for 10 min at 4°C, the DNA pellet was washed with 70%
ethanol, air-dried and finally the purified DNA pellet was dissolved in 50 μl of milliQ
(MQ)/water.
20
Example 4: PCR analysis for detection of viral DNA. For detecting viral DNA, all the preliminary PCR amplifications were performed by
using PhusionTM High fidelity DNA polymerase (Thermo Scientific, USA). The
25 composition of 20 μl PCR mix used for amplification included the following
components: 50-100 ng of genomic DNA as template, 1X HF reaction buffer, 200
μM dNTPs, 0.2 μM forward primer, 0.2 μM reverse primer and one unit of Phusion
polymerase. The PCR analysis was done using primers (SEQ ID 5 and SEQ ID 6)
18
that amplify the coat-protein gene fragment of OELCuV DNA. Special set of abutting
primers (SEQ ID 7 and SEQ ID 8) were designed from the OELCuV genome to
ensure the viral replicational release of 2.7 kb fragments by PCR. DNA from mockinoculated plants was used as negative control. Since C4 ORF is the most variable
5 region in okra viruses, primers amplifying the C4 region of OELCuV (SEQ ID 9 and
SEQ ID 10) were used to confirm the detection of OELCuV being agroinoculated. To
detect BYVMB, primers shown in SEQ ID 11 and SEQ ID 12 were used.
Example 5: DNA dot-blot analysis.
10
Dot-blot analysis was performed to study the accumulation of viral DNA in
agroinoculated okra plants. Total genomic DNA was isolated from agroinoculated
plants at 22 dpi using the method described in Example 3. For Dot-blot analysis, DIG
High Prime DNA Labeling and Detection Starter Kit II (Roche) was used as
15 described below. The probe for hybridization was derived from a 770 bp coat protein
gene fragment of OELCuV (SEQ ID NO. 1). One μg of DNA was spotted for each of
the inoculated and mock-inoculated samples on nylon membrane (Amersham
Hybond-N+, GE Healthcare, USA). As a positive control, 25 ng of OELCuV full
length cloned plasmid DNA was also spotted onto the membrane. The membrane was
20 then placed on Whatman 3MM-paper soaked with 10× SSC for UV cross-linking.
After the cross-linking, the membrane was briefly rinsed in double-distilled water and
allowed to air-dry. For hybridisation, an appropriate volume of DIG Easy Hyb buffer
(10 ml/100 cm2
filter) was pre-heated to hybridization temperature (48°C). The
membrane was pre-hybridized for 30 min with gentle agitation in an appropriate
25 container. After that, DIG-labeled DNA probe (about 25 ng/ml DIG Easy Hyb buffer)
was denatured by boiling for 5 min and rapidly cooling in ice/water. The denatured
DIG-labeled DNA probe was then added to pre-heated DIG Easy Hyb buffer (3.5
ml/100 cm2 membrane) and mixed well by avoiding foaming. The prehybridization
19
solution was poured off and probe/hybridization mixture was added to the nylon
membrane. It was then followed by incubation for 10-12 hours with gentle agitation.
After overnight incubation, the membrane was then washed twice with 2 × SSC,
0.1% SDS at 37°C under constant agitation for 5 minutes. It was then followed by
5 washing stringently with 0.5 × SSC, 0.1% SDS (prewarmed to wash temperature) at
65°C under constant agitation twice for 15 minutes. All incubations after that were
performed at 37°C with agitation. After hybridization and stringency washes, the
membrane was rinsed briefly (1-5) min in the Washing buffer [0.1 M maleic acid,
0.15 M NaCl; pH 7.5 (20°C); 0.3% (v/v) Tween 20]. Then, incubated for 30 min in
10 100 ml Blocking solution [1 × working solution was prepared by diluting 10 ×
Blocking solution 1:10 with maleic acid buffer {0.1 M Maleic acid, 0.15 M NaCl;
adjust with NaOH (solid) to pH 7.5 (20°C)}]. Incubation with 20 ml Antibody
solution (containing Anti-DIG antibody, supplied in the kit) for 30 minutes was
performed followed by washing with 100 ml of Washing buffer for 15 minutes. The
15 washing step was repeated again to get rid of extra antibody solution.
For detection of hybridization, the membrane was equilibrated for 2-5 min in 20 ml
Detection Buffer [0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20°C)]. The nylon membrane
was placed with DNA side facing up on a development folder (or hybridization bag)
and 1 ml CSPD ready-to-use (provided in kit) was applied. The membrane was
20 immediately covered with the second sheet of the folder to spread the substrate
evenly to avoid air bubbles over the membrane. The excess liquid was squeezed out
and the edges of the development folder were sealed. The membrane was incubated
for 10 min at 37°C to enhance the luminescent reaction. The signals indicating
hybridization of the probe were detected by exposing the membrane using X-ray film
25 in dark for 5 min at 37°C. Example 6: Growing of okra plants
20
Seeds of okra were sown directly in pots containing soil and kept at 28-30°C for
germination. After true leaves emerged, plants were shifted to bigger pots and kept in
the greenhouse at 30-32°C and 100% humidity, with 16 hours’ light and 8 hours’ dark cycle, the other parameters remaining the same.
5
Now, the crux of the invention is claimed implicitly and explicitly through the
following claims. Each of the appended claims defines a separate invention, which
for infringement purposes is recognized as including equivalents to the various
elements or limitations specified in the claims. Depending on the context, all
10 references below to the “invention” may in some cases refer to certain specific
embodiments only. In other cases, it will be recognized that references to the
“invention” will refer to subject matter recited in one or more, but not necessarily all,
of the claims. Groupings of alternative elements or embodiments of the invention
disclosed herein are not to be construed as limitations. Each group member can be
15 referred to a claimed individually or in any combination with other members of the
group or other elements found herein. One or more members of a group can be
included in or deleted from, a group for reasons of convenience and/ or patentability.
When any such inclusion or deletion occurs, the specification is herein deemed to
contain the group as modified thus fulfilling the written description of all groups used
20 in the appended claims.

WE CLAIM:

1. The DNA constructs comprising partial dimer derived from OELCuV and
BYVMB respectively disclosed under SEQ ID No.: 2 and SEQ ID No.: 14. 5
2. The primer pairs for the DNA constructs as claimed in Claim 1, wherein said
primer sets consisting of SEQ ID No.: 3, SEQ ID No.: 4, SEQ ID No.: 5, SEQ
ID No.: 6, SEQ ID No.: 7, SEQ ID No.: 8, SEQ ID No.: 9, SEQ ID No.: 10,
SEQ ID No.: 11, SEQ ID No.: 12, SEQ ID No.: 15, and SEQ ID No.: 16.
10
3. The DNA constructs as claimed in Claim 1, wherein the viral genomes are
derived from a geminivirus and associated betasatellite respectively.
4. The DNA constructs as claimed in Claim 1, wherein the viral and satellite
15 genomes comprise nucleotide sequences of GenBank accession numbers
KJ462076 and KJ462074.
5. The method of agroinoculation of DNA constructs as claimed in Claim 1, in a
plant selected from the family Malvaceae, preferably okra (Abelmoschus
20 esculentus).
6. A method of producing viral infection in okra plant, comprising the steps:
introducing the DNA constructs as claimed in Claim 1 into an okra plant and
growing the plant under conditions which result in symptoms of petiole
25 bending, yellowing, leaf curling and profuse branching.
7. The method as claimed in Claim 6, wherein the step of introducing the DNA
into plants is agroinoculation.
8. The method as claimed in Claim 6, wherein the step of introducing the
agroinfectious construct is selected from the group consisting of particle
bombardment, agrobacterium-mediated transformation, agrodrench, abrasion
5 of plant surfaces and plasmid inoculation.