ABSTRACT Thirty interspecific hybridizations of inter-or intra-section of Dendrobium were made. Five crosses showed good compatibility and could yield hybrids as follows: 2 intersectional hybridizations, 1) section Phalaenanthe: Den. phalaenopsis (D017) × section Formosae: Den. draconis (D022) and 2) section Dendrobium: Den. finlayanum (D030) × section Formosae: Den. cariniferum (D018); and 3 intrasectional hybridizations, 1) section Dendrobium: D030 × Den. nobile (D031), 2) section Formosae: Den. trigonopus (D037) × D022, and 3) D037 × Den. infundibulum (D034). The greatest number of plantlets (1,250 plantlets) was obtained from cross D037 × D022 whereas the least number (3 plantlets) was found in cross D037 × D034. After transplanting for 6 months, survival rate of D037 × D034 was 100% whereas no survival plant was found in cross D030 × D018. Parents and hybrids of 5 crosses were analyzed by RAPD using 21 decamer primers and the number of primers generating polymorphic patterns of each cross varied. RAPD markers were able to differentiate parents in inter-and intra-sectional crosses and these markers could confirm the interspecific hybrids

Nuttha Kuanprasert

Pimchai Apavatjrut

Sirilak Inthawong

Weenun Bundithya

CiteSeerX

Kasetsart J. (Nat. Sci.) 40 : 456 - 461 (2006)
Analysis of Intersectional Hybrids of  Dendrobium by RAPD Technique
Sirilak Inthawong1*, Weenun Bundithya2, Nuttha Kuanprasert 2
and Pimchai Apavatjrut2
ABSTRACT
 Thirty interspecific hybridizations of inter- or intra-section of  Dendrobium were made. Five
crosses showed good compatibility and could yield hybrids as follows: 2 intersectional hybridizations,
1) section Phalaenanthe: Den. phalaenopsis (D017) × section Formosae:  Den. draconis (D022) and
2) section Dendrobium: Den. finlayanum (D030) × section Formosae: Den. cariniferum (D018); and
3 intrasectional hybridizations, 1) section Dendrobium: D030 × Den. nobile (D031), 2) section Formosae:
Den. trigonopus (D037) × D022, and 3) D037 × Den. infundibulum (D034). The greatest number of
plantlets (1,250 plantlets) was obtained from cross D037 × D022 whereas the least number (3 plantlets)
was found in cross D037 × D034. After transplanting for 6 months, survival rate of D037 × D034 was
100% whereas no survival plant was found in cross D030 × D018.
Parents and hybrids of 5 crosses were analyzed by RAPD using 21 decamer primers and the
number of primers generating polymorphic patterns of each cross varied. RAPD markers were able to
differentiate parents in inter- and intra-sectional crosses and these markers could confirm the interspecific
hybrids.
Key words: Dendrobium,  interspecific hybridization, RAPD technique
1 Development of Economic Flower Crop : Orchid (DEFCO) Project, Faculty of Agriculture, Chiang Mai University, Chiangmai
50200, Thailand.
2 Department of Horticulture, Faculty of Agriculture, Chiang Mai University, Chiangmai  50200, Thailand.
* Corresponding author, e-mail: Jupiter496@yahoo.com
INTRODUCTION
Dendrobium is one of the largest genera
in the family Orchidaceae. It comprises over 1,000
species distributed from the foothills of the
Himalayas through Southeast Asia to Japan,
Australia and the Pacific Islands. Botanists divided
this genus into 41 sections (Baker et al., 1996).
About 80% of world’s dendrobium
production is from Thailand. Commercial growers
put major efforts on breeding for novel
Dendrobiums with desirable traits. Traditional
breeding is limited by time and cross-
incompatibility among inter- and intra-section
(Chia et al., 2001). From an interesting study of
sexual compatibility in Dendrobium, Wilfret and
Kamemoto (1969) made a systematic study
utilizing 37 species in 10 of Schlecter (1912) 41
sections for 721 pollinations, 5 intrasectional and
20 intersectional combinations resulted in
successful hybrids. By making crosses among 8
horticultural important sections, Kamemoto and
Wilfret (1980) could predict obtainable hybrid
percentage and observe relationship among those
species. Furthermore, Kamemoto (1987) found
possible intersectional crosses from 4 sections,
Received date : 16/07/05 Accepted date : 30/01/06
Phalaenanthe, Ceratobium, Eleutheroglossum
and Latourea. This finding has given numerous
impact on dendrobium breeding program,
especially for commercial potted cultivars.
However, novel characteristics in terms of shapes,
colors, growth and yields are still desirable.
Several Thai Dendrobium orchid species in
sections Callista, Dendrobium and Formosae
show some unique characteristics for example
sweet scent of Den. scabrilinque, glossy and
fragrant flower of Den. trigonopus, bright orange
color of Den. unicum and compact plant and
attractive lip of Den. cruentum. They are good
sources for such special characters that can
possibly be introduced to new commercial hybrids.
The development of molecular markers
for identification of genotypes and early detection
of inter-specific hybrids may be important
for breeding programs and in protecting
plant breeders’ rights (Benedetti et al., 1998).
RAPD (Random Amplified Polymorphic DNA)
technique provides a useful tool for breeding
applications. This technique followed a simple and
fast procedure, required a low quantity of DNA
and was easily automated (Ballester and Vicent,
1998).
In some recent publications, RAPD
technique has been used widely for identification
of interspecific hybrids in several kinds of plants,
such as Cattleya (Benner et al., 1995),
Phalaenopsis (Chen et al., 2001), Anthurium
(Ranamukhaarachchi et al., 2001), Lilium
(Wiejacha et al., 2001), asparagus (Caporali et al.,
1996), pepper (Balleter and Vicente, 1998), carrot
(Grzebelus et al., 2001), blueberry (Levi and
Rowland, 1997), almond (Bartolozzi et al., 1998)
and pear (Sharifani and Jackson, 2000).
In this study, RAPD technique was used
to trace some markers among inter- or intra-
sectional hybrids of Dendrobium and their parental
lines.
MATERIALS AND METHODS
Plant materials
Eleven species of Thai Dendrobium
orchid were obtained from 4 sections, 1) section
Callista: Den. chrysotoxum (D026), 2) section
Dendrobium: Den. crystallinum (D044), Den.
findlayanum (D030), Den. nobile (D031) and Den.
unicum (D015), 3) section Formosae: Den.
cariniferum (D018), Den. draconis (D022), Den.
infundibulum (D034), Den. scabrilinque (D012)
and Den. trigonopus (D037) and 4) section
Phalaenanthe: Den. phalaenopsis (D017).
Testing for cross compatibility
Thirty interspecific hybridizations of
inter- or intra-section were studied. The result of
cross compatibility and number of capsules were
recorded. Three months after pollination, capsule
of each cross was harvested and then germinated
in vitro using Vacin and Went (1949) agar medium.
A number of plantlets and survival rate after 6-
month transplanting were recorded.
RAPD analysis
DNA was extracted from young leaves
of 5 selected hybrids and their parents using a 2x
CTAB (hexadecyltrimethyl ammonium bromide)
procedure (Doyle and Doyle, 1990). Polymerase
chain reaction (PCR) was carried out in a 20 µl
reaction mixture containing 5 ng of DNA template,
1x PCR buffer (20mM Tris-HCl pH 8.0, 0.1mM
EDTA, 1mM DTT, 50% glycerol), 1.5 mM MgCl2,
100 µM dNTPs, 100 ng primer, 0.8 unit Taq DNA
polymerase and dH2O. Twenty-one decamer
primers, OPF01-20 and OPD03 (Operon
Technologies Inc.) were used for PCR
amplification. The DNA was amplified in the
Perkin Elmer Gene Amp PCR System 2400
(Perkin-Elmer Cetus Co.). The PCR program was
modified from Chen et al. (1998) as follows: 2
cycles of 94 °C 60 s, 36 °C 10 s and 72 °C 70 s; 30
cycles of 94 °C 60 s, 42 °C 45 s and 72 °C 70 s;
Kasetsart J. (Nat. Sci.) 40(2) 457
Kasetsart J. (Nat. Sci.) 40(2)458
and 1 cycle at 72 °C for 240 s. The PCR products
were kept at 4 °C prior to analysis.
The PCR products were separated by
electrophoresis on 1.8% agarose gels in 1x TBE
buffer. The amplified DNA bands were stained in
0.1 µg/ml ethidium bromide and then
photographed under UV light using Gel
Documentation (Lab Focus. Co., Ltd.).
RESULTS
Testing for cross compatibility
Thirty interspecific hybridizations, 21
intersectional and 9 intrasectional crosses were
made. Compatibility was found and capsules were
set in the following crosses: 2 intersectional
hybridizations (9.52%), 1) section Phalaenanthe:
Den. phalaenopsis (D017) × section Formosae:
Den. draconis (D022) and 2) section Dendrobium:
Den. findlayanum (D030) × section Formosae:
Den. cariniferum (D018) and 3 intrasectional
hybridizations (33.3%), 1) section Dendrobium:
D030 × Den. nobile (D031), 2) section Formosae:
Den. trigonopus (D037) × D022 and 3) D037 ×
Den. infundibulum (D034). After culturing in
modified Vacin and Went (1949)(CMU1) agar
medium for 8 months, the greatest number of
hybrid seedlings was 1,250 plantlets obtained from
D037 × D022 while other crosses, D030 × D031,
D017 × D022, D030 × D018, and D037 × D034,
showed various numbers of hybrid seedlings,
i.e. 600, 125, 100, and 3 plantlets, respectively
(Table 1).
Eight-month old hybrid seedlings were
transplanted from in vitro to shadehouse condition.
After six-month period, seedlings of D037 × D034
showed the greatest survival rate (100%) whereas
those of crosses  D017 × D022, D030 × D031 and
D037 × D022 yielded only 55.6, 26.7 and 19.4%
survival rate, respectively. Hybrid seedlings of
crosses D030 × D018 were not viable (Table 1).
RAPD analysis
The 21 decamer primers were evaluated
for amplification of 4 compatible crosses and their
hybrids. The number of primers giving
polymorphic pattern varied among crosses.
Suitable primers for each cross could be described
as follows: 7 primers (OPF01, 02, 03, 04, 05, 06
and OPD03) for cross D017 × D022; 6 primers
(OPF01, 02, 03, 04, 06 and 20) for cross D037 ×
D022; 5 primers (OPF01, 02, 04, 05 and 14) for
cross D030 × D031; and 5 primers (OPF01, 04,
06, 14 and OPD03) for cross D037 × D034.
Selected DNA fingerprints (Figure 1-4) were
presented showing polymorphic RAPD markers
from either parents that appeared in hybrid
banding. Two markers from cross D017 × D022,
1040 bp. and 739 bp., represented those derived
from D017 (female parent) and D022 (male parent)
respectively (Figure 1). For cross D037 × D022,
the 1239 bp. and 1219 bp. from D037 (female
Table 1 Number of seedlings per pod obtained in vitro, Number of transplanted seedlings and Number
of survival plantlets after 6-month transplanting.
Crosses No. of seedlings per pod No. of transplanted No. of survival plantlets after
obtained in vitro seedlings 6-month transplanting
D017  × D022 125 18 10
D018  × D030 no viable seedling - -
D030  × D018 100 60 0
D030  × D031 600 390 104
D037  × D034 3 3 3
D037  × D022 1,250 350 68
D034  × D022 no viable seedling - -
Kasetsart J. (Nat. Sci.) 40(2) 459
parent) and D022 (male parent), respectively,
appeared in hybrids (Figure 2). Three markers
were found in cross D030 × D031 of which the
1182 bp. marker was derived from D030 (female
parent) while the 831 bp. and 750 bp. derived from
D031 (male parent) (Figure 3). Four markers were
found in cross D037 × D034 of which the 564 bp.,
490 bp. and 273 bp. Markers was derived from
D037 (female parent) while the 476 bp. from D034
(male parent) (Figure 4).
DISCUSSION
Testing for cross compatibility
Thirty interspecific hybridizations of
inter- or intra-section were done. Two
intersectional hybridizations, D017 × D022 and
D030 × D018; and 3 intrasectional hybridizations,
D030 × D031, D037 × D022 and D037 × D034
showed good compatibility and could yield
hybrids but in the other crosses were
incompatibility. Wilfret and Kamemoto (1969)
reported that failure in producing successful intra-
and inter-sectional crosses could show some
Figure 1 RAPD banding pattern of cross  D017
× D022 generated by primer OPF03:
M: 50-2500 bp. marker; P1: D017; P2:
D022 and 1-5: F1 hybrids. 1014 and 736
bp. are RAPD markers.
Figure 2 RAPD banding pattern of cross  D037
× D022 generated by primer OPF03:
M: 50-2500 bp. marker; P1: D037; P2:
D022 and 1-5: F1 hybrids. 1239 and
1219 bp. are RAPD markers.
Figure 3 RAPD banding pattern of cross  D030
× D031 generated by primer OPF14:
M: 50-2500 bp. marker; P1: D037; P2:
D022 and 1-5: F1 hybrids. 1182, 831
and 750 bp. are RAPD markers.
Figure 4 RAPD banding pattern of cross  D037
× D034 generated by primer OPF06:
M: 50-2500 bp. marker; P1: D037; P2:
D034 and 1-3: F1 hybrids. 564, 490, 476
and 273 bp. are RAPD markers.
Kasetsart J. (Nat. Sci.) 40(2)460
degrees of relationship of species, success in
obtaining interspecific hybrids usually indicated
a close relationship. One of the main problems in
making new hybrid cultivar in Dendrobium was
difficulty of fruit setting. In this study, when
reciprocal crosses were made of all compatible
crosses, it was found that none of them could
produce hybrid. It showed that certain species
could be used as only mother plant and could not
be used as pollen donor. This is similar to report
by Reed et al. (2002) in Clethra that reciprocal
crosses failed to set fruit.  Wilfret and Kamemoto
(1969) reported that the Dendrobium breeding
program needs time to test the compatibility of
interspecific crosses and study on the factors of
incompatible crosses.
RAPD analysis
Confirmation of the interspecific hybrid
relied on molecular comparisons of the hybrids
with their parents (Hansen, 1998). Twenty-one
decamer primers were used for amplification of
the hybrids which were obtained in this study and
their parents, and the number of primers giving
polymorphic patterns varied among crosses. Two
of these primers namely OPF01 and OPF04 (data
not shown) could produce different DNA
fingerprint patterns that enabled differentiation
among the parental cultivars of all intrasectional
crosses. It indicated that any Dendrobium cultivars
had a unique genetics (Ranamukhaarachchi et al.,
2001).
It was able to prove that the hybrids were
the result of genetic combination of their parents.
RAPD bandings of hybrids when using suitable
primers for each cross, clearly indicated that the
DNA came from the 2 parents by the appearance
of RAPD markers i.e. OPF03736, 1040 in cross
D017 × D022, OPF031219, 1239 in cross D037 ×
D022, OPF14750, 831, 1182 in cross D030 × D031
and OPF06273, 476, 490, 564  in cross D037 × D034.
The same results were also reported in the other
orchids, such as Cattleya by Benner et al. (1995)
and Phalaenopsis by Chen et al. (2001).
The establishment of polymorphic bands
in the parental genotypes of Dendrobium was
relatively easy, although not all of the markers
were useful for the detection of hybrids. Selected
bands could be used for further hybridizations. Due
to high heterozygosity of the cultivars, some
polymorphic bands could not be used for hybrid
identification (Wiejacha et al., 2001).
In summary, RAPD marker could be
used for identification of Dendrobium cultivars as
shown by different DNA fingerprinting from each
plant. These polymorphisms could be used for
detection of inter- or intra-sectional hybrid.
ACKNOWLEDGEMENTS
This work was financially supported by
the Development of Economic Flower Crop :
Orchid (DEFCO) Project, Faculty of Agriculture,
Chiang Mai University. We thank for the use of
equipments from ‘ H.R.H. Princess Maha Chakri
Sirindhorn Plant Germplasm Project’ ( RSPG
Project), Chiang Mai, Thailand.
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Primer code Sequence (5′ - 3′)
OPF01 ACGGATCCTG
OPF02 GAGGATCCCT
OPF03 CCTGATCACC
OPF04 GGTGATCAGG
OPF05 CCGAATTCCC
OPF06 GGGAATTCCC
OPF14 TGCTGCAGGT
OPF20 GGTCTAGAGG
OPD03 GTCGCCGTCA
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Analysis of Intersectional Hybrids of Dendrobium by RAPD Technique

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Analysis of Intersectional Hybrids of Dendrobium by RAPD Technique

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