Scientia Horticulturae 161 (2013) 357360

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Scientia Horticulturae
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Intergeneric hybridization in papaya for PRSV tolerance
M.R. Dinesh
a,
, G.L. Veena
b
, C. Vasugi
a
, M. Krishna Reddy
a
, K.V. Ravishankar
a
a
Indian Institute of Horticultural Research, Hessarghatta Lake Post, Bangalore 560089, India
b
Division of Pomology, Post Graduate School, University of Horticultural Sciences, Bangalore, India
a r t i c l e i n f o
Article history:
Received 17 January 2013
Received in revised form2 July 2013
Accepted 5 July 2013
Keywords:
Breeding
Intergeneric hybridization
Papaya
Primers
PRSV
Selng and sibmating
a b s t r a c t
Papaya ringspot virus is one of the major impediments in papaya cultivation. Intergeneric crossing
carried out using the PRSV resistant wild species Vasconcellea cauliora as male parent, with the papaya
variety Arka Surya as female parent resulted in the intergeneric hybrid progenies. The progenies found
resistant after articial inoculation were eld planted. The progenies segregated for castor and papaya
type leaves with fruits having yellow and pink pulp. The tolerant hermaphrodite progenies selected based
onthe fruit characteristics were selfed. Five generations of single plant selection was carried out by selng
the hermaphrodite tolerant progenies having castor type leaf. The hybrids in the sixth generation were
validated using ISSR markers. The banding pattern was obtained with the primers UBC-815, UBC-834,
UBC-836, UBC-841 and UBC-844 showed that they are intergeneric hybrids.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Papaya (Carica papaya L.) is one of the important fruit crops that
are being grown commercially in India. One of the main impedi-
ments in its cultivation is the infestation of Papaya ringspot virus
(PRSV), which is a major limiting factor in papaya production. The
virus causes diverse symptoms that include vein clearing, mottling,
malformed leaves, liformy, ring spots and streaks on fruits, stem
and petioles and stunting. The virus was rst recorded in from
western regions of India in 1958 (Capoor and Varma, 1958) and
since then it has spread throughout the country. A breeding pro-
gramme using this source in Florida (USA) resulted in the release
of Cariora, a dioecious cultivar with moderate resistance and
fruit quality (Conover et al., 1986). The virus isolates have been
bio-typed to papaya infecting (Type P) and non-papaya infecting
(Type W) types. PRSV belongs to the poty virus group of plant
viruses and is transmitted by several species of aphids in a non-
persistent manner (Purcifull et al., 1984). All the known varieties of
papaya are susceptible to this disease. However, some of the wild
species like Vasconcellea cauliora have been found to be resistant
to this disease (Jimenez and Horovitz, 1958; Horovitz and Jimenez,
1967). Work carried out earlier has shown that use of Sucrose
resulted in viable seed set from the intergeneric hybridization
(Dinesh et al., 2007). The intergeneric hybridization was carried

Corresponding author. Tel.: +91 809448064198.

E-mail addresses: drmrdinesh@gmail.com, drmrdinesh@yahoo.co.in
(M.R. Dinesh).
out with V. cauliora as male parent with Arka Surya as female
parent. The objective of the study was to work out a methodology
for developing PRSV tolerant intergeneric progeny by single plant
selection and selng of hermaphrodites and validating these prog-
enies in successive generations by ISSR markers and by screening
for tolerance/resistant in every generation.
2. Materials and methods
The C. papaya var Arka Surya was used as the female parent for
intergeneric crossing. It is a gynodioecious variety bearing medium
sized fruits of 600800g with pink pulp colour and high TSS of
1314

Brix. It was derived fromthe parentage Sunrise SoloPink

FleshSweet. The male parent V. cauliora is a dioecious wildspecies
having castor type leaf and producing non-edible fruits with yel-
low pulp. Crossing was carried out in the morning hours between
9 and 11a.m. The pollen fertility and viability were conrmed
using Alexanders stain (Alexander, 1987). The stigma of the female
parent was smeared with Sucrose (5%) and then the pollen was
smeared on top of that. On an average 50 crosses were made per
plant and the fruits were harvested at the right stage of maturity
and seeds were extracted.
The virus inoculum was prepared by homogenizing infected
papaya leaves in 0.1mol/L phosphate buffer, pH 7, and applied
by gentle rubbing onto carborundum-dusted leaves. Plants were
inoculated three times at two week intervals. Assessment of symp-
toms was done one month after the nal inoculation and plants
were assayed for virus concentration by ELISA as per the protocol
developed (Magdalita et al., 1998).
0304-4238/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scienta.2013.07.009
358 M.R. Dinesh et al. / Scientia Horticulturae 161 (2013) 357360
Arka Surya x V. cauliflora
(Sunrise Solo x Pink Flesh Sweet)

F
1
progenies from the cross
(Segregation ratio of 3:1 for papaya to castor)
Screening in the laboratory for PRSV
Field planting of the resistant progenies---2 sets one with castor leaf and another with
papaya leaf
Sibmate the progenies and raise the next generation (S
1
)

Screen the progenies for PRSV and evaluate them for fruit characteristics

Sibmate the progenies and raise the next generation (S
2
)(Single plant selections)

Repeat the operation for another five cycles with two sets of progenies
Selection of progenies with desirable fruit characteristics and PRSV tolerance
Fig. 1. Schematic diagram.
Hybridity conrmationthroughmolecular characterizationwas
done by extracting the genomic DNA using CTAB method (Lodhi
et al., 1994) yielded good amount of DNA. DNA recovery varied
from750ng/l to 3640.25ng/l. The ratio of DNAto protein ranged
from1.85 to 3.08. 25l of total reactionmixture was prepared each
by adding 2.5l of reaction buffer, 5l of primer, 2.5l of DNTPs,
Taq DNApolymerase of 0.25l along with 9.75l of water and 5l
of template DNA. PCR conditions were optimized for informative
and reproducible nger print prole. Ten ISSR primers (viz., UBC
807, 810, 814, 815, 817, 834, 836, 841, 844, 856) were used for
validating.
3. Results and discussion
The schematic diagramfor the procedure adopted for develop-
ing progenies fromthe intergeneric crosses is given in Fig. 1.
As early as in 1957, the interspecic crossing relationship in
papaya was studied (Sawant, 1958). Interspecic hybridization
generated a hybrid progeny tolerant to PRSV, however, on back-
crossing tolerance was lost (Iyer et al., 1987).
The methodology (Fig. 1), mentioned has been adopted to
developintergeneric lines tolerant toPRSV. Inthis procedure, back-
crossing has not been carried out and only sibmating and selection
has been carried out depending on the quality of the fruits by sib-
mating. The intergeneric hybridization carried out by the use of
5% Sucrose, resulted in a seed set of 13.1% per fruit fromwhich 10
seedlings were raised (Dinesh et al., 2007). The procedure shows
that in the F
1
generation there is segregation in the ratio of 3:1
for papaya to castor type plants. The two sets of population gen-
erated thus, one with castor leaf type and another with papaya
leaf type were maintained by selng the hermaphrodites. The suc-
cessive generations thus raised were subjected to screening and
the tolerant one taken to the eld (Figs. 2 and 3). The progeny
Fig. 2. Hybridization using the female and male parents.
M.R. Dinesh et al. / Scientia Horticulturae 161 (2013) 357360 359
Fig. 3. Single plant of intergeneric hybrid.
population raised showed stability for plant and fruit character-
istics by the fourth generation. These were sibmated to raise the
fth generation. In a study carried out among many varieties only
three entries survived to the end of the 2-year trial: Sun Up, Rain-
bowand Dagua Yellow, the rst 2 (transgenic fromHawaii) tested
PRV negative by ELISA, while Dagua Yellowtested positive. In spite
of that, this papaya exhibited only mild virus symptoms and con-
tinued to produce fruit (Wall and Wiecko, 2012). The breeding
programme chalkedout by themto improve Dagua Yellowby elim-
inating the male character and change its pulp colour fromyellow
to red resulted in the F
4
generation seed of PRSV-tolerant Dagua
Yellow, Orange and Red. Inbred lines consisting of S
5
to S
9
lines
developedby continuous self andsibpollinationwere obtainedand
these inbred lines were then selected for specic traits including:
hightotal soluble solids, highpapainyield, prolic bearing, tolerant
to PRSV and big-fruited (Magdalita Villegas et al., 1988). This study
shows similar results to that of the present one indicating that by
sibmating and selection in segregating population hermaphrodite
progenies could be obtained. There is always a distinct advantage
in choosing gynodioecious variety as a female parent over the dioe-
cious variety since there is a need to look for two resistant plants
in male and female in the case of dioecious varieties. However, as
proposed in this study, single plant selection can be practiced in
gynodioecious varieties by selng the hermaphrodites.
The RCBD analysis (Table 1) carried out for selected progenies
showed that there was signicant difference among the proge-
nies for different fruit characteristics. One interesting feature is
the segregation for yellow and pink colour in the pulp. One of the
advantages in any breeding programme is the presence of morpho-
logical markers for a conventional breeder. Along with the pulp
colour, in this case yellow colour, which is inherited from the
cauliora, it is the presence of castor type leaf, which gives indi-
cation of the morphological marker conclusively proving that the
hybrids are fromthe intergeneric crossing. Further sibmating of the
progenies for pulp colour has resulted in the stabilization of these
characters in the progenies. It is also to be noted here that sin-
gle plant selection of hermaphrodite types is very much reliable as
fertile hermaphrodites selected and selfed would result in a stable
population, just as sibmating it with the females.
Development of successful PRSV tolerant line by embryo res-
cue from the intergeneric cross between papaya and Vasconcellea
quercifolia has also been reported (Siar et al., 2011). Further, it was
reportedthat the inoculationof crosses withPRSV-P showed60%of
theplants werenot infected, whichis alsoconrmedthroughELISA,
where as 25% of the plants showed infection with mild symptoms.
In the resistant type of plants, ELISA values were less than twice
those of non-inoculated plants. However, in this study embryo res-
cue was not attempted and the progenies were raised by seeds
obtained fromthe intergeneric crossing.
3.1. Validation of hybridity using ISSR markers
Although, the morphological markers showed clearly that the
progenies are intergeneric hybrids, conrmation of hybridity was
carried out using ve ISSR primers primer, viz., UBC-815, UBC-834,
UBC-836 UBC-841 and UBC-844 were used. It has been shown that
the ISSR markers are better in differentiating the parents and con-
rmthe hybridity and it is known to be a better marker than other
dominant markers such as RAPD.
The banding pattern obtained with the primers UBC-815, UBC-
834, UBC-836 UBC-841 and UBC-844 showed the hybrid-nature in
progenies tested. The hybrid nature of all the 45 progenies was
marked by UBC-834 (Fig. 4). In UBC-841, unique banding pattern
was observedinV. cauliora(male parent), whereinve bands were
observed to be prominent. These bands when compared with C.
papaya showed distinct nature with absence of rst and third band
in female parent. In the case of UBC-841, six bands were observed
in male parent in which third, fth and sixth were distinct from
female parent and sixth band was present in all the hybrids, but
rst and fourth faint bands were observed (Fig. 4).
In a study using intergeneric hybrids of C. papaya V. quercifo-
lia validated for hybridity, showed that the intergeneric hybrid was
foundtobe resistant topapaya ringspot virus similar toV. quercifo-
lia (Mendoza-Garces et al., 2010). They observed one slowmoving
Fig. 4. Polymorphic bands observed in intergeneric hybrids using ISSR: UBC 834. PCR amplied products showing polymorphismusing ISSR primer UBC 834: 1 and M 1kb
ladder; 2 A. Surya; 3 V. cauliora; 47 F1s; and 846 intergeneric hybrids.
360 M.R. Dinesh et al. / Scientia Horticulturae 161 (2013) 357360
Table 1
Fruit characteristics of selected intergeneric progenies.
S. No Progeny Fruit wt. (g) Fruit length (cm) Fruit width (cm) Fruit cavity index (%) Pulp thickness (cm) TSS (

Brix) Pulp colour

1 R
1
P
2
891.97 13.70 11.60 25.03 3.33 10.80 Yellowish orange
2 R
1
P
4
1477.40 15.67 14.80 31.49 3.37 7.77 Yellow
3 R
2
P
1
1373.90 15.00 14.47 29.24 3.03 12.17 Deep pink
4 R
2
P
2
1492.53 15.70 14.43 22.97 3.53 11.17 Deep pink
5 R
3
P
3
896.90 13.27 12.60 28.43 2.60 10.20 Golden yellow
6 R
3
P
4
382.27 11.17 8.20 27.82 2.37 12.73 Dark yellow
7 R
3
P
9
840.10 12.77 11.40 35.26 2.33 10.70 Deep pink
SEM 118.43 0.67 0.84 3.74 0.25 1.07
CV% 19.52 8.43 11.66 20.09 14.75 17.28
band identical to the slow moving band of C. papaya and another
band identical to the single band of V. quercifolia. Since the SSR is
specic toCarica sp. andare codominant markers, they revealedthe
hybridity clearly by showing clear bright bands (Mendoza-Garces
et al., 2010). This is in conformity with the validation carried out
here, although, the V. cauliora is used here.
The method proposed and adopted here in the development
of obtaining viable and stable population by conventional breed-
ing using single plant selections/sibmating would result in tolerant
lines to PRSV. This is easy in papaya, as fertile hermaphrodites
can be chosen for selng. However, development of initial lines
would depend on the number of progenies obtained in the F
1
gen-
eration. Use of molecular markers would help in the conrmation
of hybridity.
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