See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/256092815

Pollen meiotic behavior in relation to Phalaenopsis breeding

Article  in  Acta Horticulturae · October 2010

DOI: 10.17660/ActaHortic.2010.878.15

CITATIONS READS

3 23,852

5 authors, including:

S. W. Chin Fure-Chyi Chen

National Pingtung University of Science and Technology National Pingtung University of Science and Technology
61 PUBLICATIONS   248 CITATIONS    116 PUBLICATIONS   663 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Advocacy of Science and Technology in Latin America View project

Role of AGO4 orthologs in ovule development in Carica papaya View project

All content following this page was uploaded by Pablo Bolaños-Villegas on 30 May 2014.

The user has requested enhancement of the downloaded file.

Pollen Meiotic Behavior in Relation to Phalaenopsis Breeding
S.C. Hsu1, T.C. Cheng1, P. Bolaños-Villegas2, 3, S.W. Chin1, a and F.C. Chen1, b
1
Department of Plant Industry, National Pingtung University of Science & Technology,
Taiwan
2
Institute of Tropical Agriculture & International Cooperation, National Pingtung
University of Science & Technology, Taiwan
3
Current Address: Taiwan International Graduate Program, Molecular and Biological
Agricultural Sciences, Academia Sinica, Taipei, Taiwan / Graduate Institute of Bio-
technology, National Chung Hsing University, Taichung, Taiwan

Keywords: Doritaenopsis, mitotic inhibitors, orchids, tetrad analysis

Abstract
The breeding of Phalaenopsis orchids involves the cross-pollination of two
selected parental plants. Parents with regular meiotic behavior can often produce
seeds successfully. Currently most Phalaenopsis hybrids are obtained after inter-
specific hybridization. Unfortunately breeding barriers may occur during the meiotic
process thereby affecting fertilization events and reducing seed set. Through pollen
tetrad analysis, a correlation between successful seed set and a high tetrad percentage
was observed in both Phalaenopsis and Doritaenopsis orchids. To improve the
breeding of hybrid polyploid cultivars, mitotic inhibitors were applied to young flower
buds before pollen meiosis in order to monitor changes in the production of tetrads.
When compared to the control, a higher number of dyads were observed after treat-
ment with mitotic inhibitors. Treated pollen grains were then attached to the stigma of
female parents for self- and cross-pollination, resulting in successful fruit set in several
cases.

INTRODUCTION
Phalaenopsis breeding is important in order to obtain novel cultivars by selecting
progenies with good horticultural traits, such as new flower color, flower shape and size,
and fragrance. More than 50 species exist in their native habitats, distributed in Southern
part of Taiwan, Philippines, Indonesia, Malaysia and other nearby countries (Christenson,
2001). Its alliance genera include Doritis and Kingidium (Christenson, 2001). Many new
hybrids are registered through the Royal Horticultural Society, England, every year. Most
hybrids are produced via interspecific- and intergeneric-hybridization, with crossing
barriers encountered by breeders. One of the causes of the breeding barriers is due to
abnormal pollen meiosis which may lead to ineffective sporads (Bolaños-Villegas and
Chen, 2007; Bolaños-Villegas et al., 2008).
The meiotic behavior of some Doritaenopsis hybrids has been studied by
comparing chromosome pairing and tetrad analysis in the pollen mother cells. Higher
frequency of normal tetrad and regular bivalents correlated well with seed set
(Bolaños-Villegas and Chen, 2007; Bolaños-Villegas et al., 2008). By observing sporad
types, unreduced gametes, such as monad and dyand, might occur at low frequency. By
unreduced gametes, it means 2n chromosome number existing in single pollens, which
offers the opportunity for breeding polyploids (Ramsey and Schemske, 1998). Unreduced
gametes have been reported in many plants, such as Aranda (Lee, 1987), Phalaenopsis
(Griesbach, 1985), Spathoglottis (Teoh, 1984). The objectives of this work were to further
investigate pollen meiotic behavior of some large pink and harlequin-colored
Doritaenopsis and Phalaenopsis cultivars, and the exploitation of unreduced pollens, by
treating with mitotic inhibitors, for polyploidy breeding purpose.

a
swchin@mail.npust.edu.tw
b
fchen@mail.npust.edu.tw

Proc. Ist Int’l Orchid Symposium

Eds.: M.G. Blanchard et al. 139
Acta Hort. 878, ISHS 2010
MATERIALS AND METHODS
Nine Doritaenopsis cultivars and 3 Phalaenopsis cultivars (Fig. 1) were used for
meiotic analysis. Pollinia of full bloom flowers were collected for sporad analysis
according to previous reports (Bolaños-Villegas and Chen, 2007; Bolaños-Villegas et al.,
2008). Briefly, the pollinia were fixed with 95% ethanol-glacial acetic acid (3:1 by vol),
hydrolyzed in 1 N HCl, and Pollen mother cells (PMCs) stained with lactophenol-acid
fuchsin solution (Lim et al., 2001). The pollinia were examined under an inverted
phase-light microscope (Diaphot; Nikon).
For mitotic inhibitor treatment, colchicines (0.05, 0.10%) and trifluralin (0.09,
0.13%) were evenly dissolved in lanolin paste and applied to young flower buds. Pollinia
were collected when the flower opens. Sporad analysis was as described above.
RESULTS AND DISCUSSION
Among four harlequin-type cultivars, including Dtps. Chain Xen Mammon, Dtps.
Chian Xen Magpie, Dtps. Fusheng Pink Pearl ‘KHM2155’ and Dtps. OX Firebird, high
percentage of tetrad in the pollen mother cells (PMCs) was observed (Table 1). When
used as either female or male parents, all of them were fertile upon pollination. However,
another harlequin-type cultivar, P. Sogo Sofei, did not set seeds either as pod parent or as
pollen parent to other cultivars (data not shown), despite its high number of tetrad. This
suggests that crossing barriers operate in some harlequin-type cultivars.
Tetrad frequency is also high in other tetraploid cultivars such as Dtps. Join Angel,
Dtps. Leopard Prince, Dtps. Ney Shan Gu Niang, Dtps. Nobbys Pink Lady, P. Join Grace,
P. Sogo Yoshida (Table 1). Some micronuclei or polyads were also observed in some
cultivars (Table 1). They could set seeds after pollination by pollen donors in most cases
(data not shown). Higher tetrad frequency in the PMCs has been suggested to correlate
with higher rate of capsule setting and seed formation (Bolaños-Villegas and Chen, 2007;
Bolaños-Villegas et al., 2008).
Meiotic polyloidization may contribute to the breeding of polyploid, as
demonstrated in lily (van Tuyl et al., 2005). As odd-numbered polyploids tend to have low
fertility, it is desirable to double the chromosome sets in order to increase fertility and
thus breeding effiency (Kamemoto et al., 1999). To test this concept in Phalaenopsis, we
treated the young flower buds of both Phalaenopsis Sogo Yukidian ‘V3’ and P. Tai Lin
Redangel ‘V31’ cultivars by smearing either colchicine or trifluralin lanolin pastes on
their surface. In Sogo Yukidian ‘V3’, both mitotic inhibitors treatment increased the
frequency of dyad (Table 2), which may contain 2n-gametes, while only colchicine was
effective in increasing dyad frequency of P. Tai Lin Redangel ‘V31’ (Table 3). Trifluralin
did not change the dyad frequency; instead, more micronuclei seemed to be evoked in one
case (Table 3). Using both Phalaenopsis cultivars as model plants to manipulate dyad, we
expect to adopt some diploid and triploid Phalaenopsis cultivars for mitotic inhibitor
treatments to obtain unreduced gametes with 2n-chromosome number.
CONCLUSIONS
Regular meiosis of both Doritaenopsis and Phalaenopsis hybrids may play a key
role in their fertility (Bolaños-Villegas and Chen, 2007; Bolaños-Villegas et al., 2008).
Higher frequency of normal pollen tetrads tends to contribute seed set. In some primary
intergeneric hybrids between Doritis and Phalaenopsis, designated as Doritaenopsis, the
long and short chromosomes might pair irregularly and thus resulted with low fertility
(Bolaños-Villegas and Chen, 2007; Bolaños-Villegas et al., 2008). To overcome crossing
barrier, we have successfully induced unreduced 2n-gametes in Phalaenopsis cultivars.
This may help breeding for more advanced polyploidy hybrids by selecting elite
genotypes and treating with mitotic inhibitors.
ACKNOWLEDGEMENTS
This work was supported by grants from the Food and Agriculture Agencies,
Council of Agriculture, Taiwan (grant numbers 97AS-4.2.2-FD-Z3 and 98AS-4.2.2-
FD-Z3).

140
Literature Cited
Bolaños-Villegas, P. and Chen, F.C. 2007. Cytological identification of chromosomal
rearrangements in Doritaenopsis and Phalaenopsis. J. Intl. Coop. 2:1–11.
Bolaños-Villegas, P., Chin S.W. and Chen, F.C. 2008. Meiotic chromosome behavior and
capsule setting in Doritaenopsis hybrids. J. Amer. Soc. Hort. Sci. 133:107–116.
Christenson, E.A. 2001. Phalaenopsis—A Monograph. Timber Press, Portland, Oregon.
Griesbach, R.J. 1985. Polyploidy in Phalaenopsis orchid improvement. J. Hered.
76:74–75.
Kamemoto, H., Amore, T.D. and Kuehnle, A.R. 1999. Breeding Dendrobium Orchids in
Hawaii. Univ. of Hawaii Press, Honolulu.
Lee, Y.H. 1987. Cytology and fertility of an intergeneric orchid hybrid. J. Hered. 78:
319–322.
Ramsey, J. and Schemske, D.W. 1998. Pathways, mechanisms, and rates of polyploidy
formation in flowering plants. Ann. Rev. Ecol. Systematics 29:467–501.
Teoh, S.B. 1984. Polyploid spore formation in diploid orchid species. Genetica 63:53–59.
van Tuyl, J.M., Barba-Gonzalez, R., van Silfhout, A.A., Lim, K.-B. and Ramanna, M.S.
2005. Meiotic polyploidization in five different interspecific Lilium hybrids. Acta Hort.
673:99–105.

Tables

Table 1. Distribution of sporad type in Doritaenopsis and Phalaenopsis orchids.

PMCs
Cultivars Tetrad Tetrad Tetrad
Dyad Triad Tetrad +1 mc* +2 mc +3 mc Polyads
Dtps. Chain Xen Mammon 15 17 911 56 1 0 0
Dtps. Chian Xen Magpie 5 15 848 117 13 1 1
Dtps. Fusheng Pink Pearl ‘KHM2155’ 6 11 856 45 7 0 75
Dtps. I-Hsin New Girl ‘KHM205’ 1 10 752 203 27 1 6
Dtps. Join Angel ‘TH274’ 1 6 947 44 1 0 1
Dtps. Leopard Prince ‘F977’ 0 7 932 54 4 0 3
Dtps. Ney Shan Gu Niang 6 6 864 78 11 0 35
Dtps. Nobbys Pink Lady ‘K42305’ 3 3 948 43 3 0 0
Dtps. OX Firebird ‘OX1277’ 8 15 898 75 4 0 0
P. Join Grace ‘TH288-4’ 2 7 950 36 3 0 2
P. Sogo Sofei 0 0 680 227 41 11 41
P. Sogo Yoshida ‘F1320’ 0 6 974 20 0 0 0
The total scoring number per individual sample is 1000 pollen mother cells (PMCs).
*mc=micronuclei.

141
Table 2. Distribution of sporad type in Phalaenopsis Sogo Yukidian ‘V3’ after mitotic
inhibitor treatments.

PMCs
Chemicals
Monad Dyad Triad Tetrad Tetrad + mc*
Control 1 3 12 942 42
Colchicine 0.05% 104 141 226 521 7
Colchicine 0.10% 33 40 232 676 19
Trifluralin 0.09% 11 15 81 883 11
Trifluralin 0.13% 14 43 152 779 13
The total scoring number per individual sample is 1000 pollen mother cells (PMCs).
*
mc = micronuclei.

Table 3. Distribution of sporad type in Phalaenopsis Tai Lin Redangel ‘V31’ after mitotic
inhibitor treatments.

Chemicals/ PMCs
bud size (cm) Monad Dyad Triad Tetrad Tetrad + mc*
Control 2 7 1 785 205
Colchicine 0.05% 0 54 26 745 175
1.1/0.8**
Colchicine 0.05% 0 16 4 839 141
1.4/1.0
Colchicine 0.05% 0 3 7 776 214
1.4/1.2
Colchicine 0.05% 2 7 5 765 121
1.7/1.4***
Trifluralin 0.13% 0 4 6 757 233
1.2/0.9
Trifluralin 0.13% 5 7 8 548 432
1.5/1.1
Trifluralin 0.13% 0 9 9 780 202
1.6/1.3
Trifluralin 0.13% 0 9 6 778 207
1.7/1.4
The total scoring number per individual sample is 1,000 PMCs.
*
mc = micronuclei.
**
Size range of flower buds.
***
A total number of 900 PMCs scored.

142
Figurese

Fig. 1. Morphology of Doritaenopsis and Phalaenopsis cultivars. (A) Dtps. Leopard

Prince ‘F977’; (B) Dtps. OX Firebird ‘OX1277’; (C) Dtps. Chain Xen Mammon;
(D) Dtps. Chian Xen Magpie; (E) P. Sogo Sofei; (F) Dtps. Fusheng Pink Pearl
‘KHM2155’; (G) Dtps. Nobbys Pink Lady ‘K42305’; (H) P. Sogo Yoshida
‘F1320’; (I) Dtps. I-Hsin New Girl ‘KHM205’; (J) Dtps. Ney Shan Gu Niang; (K)
Dtps. Join Angel ‘TH274’; and (L) P. Join Grace ‘TH288-4’.

143
 144

View publication stats