Download Diploid female gametes induced by colchicine in Oriental lilies

Diploid female gametes induced by colchicine in Oriental lilies
Hongzhi Wu a, Sixiang Zheng a,b,*, Yueqiu He a, Guijun Yan c, Yufen Bi d, Youyong Zhu a
a Key
Laboratory for Plant Pathology of Yunnan Province, Yunnan Agricultural University, Kunming 650201, China
b Leisen
c School
Science and Technology Limited Company, Kunming 650011, China
of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia,
35, Stirling Highway, Crawley, WA 6009, Australia
d Department
of Pasture Science, Yunnan Agricultural University, Kunming 650201, China
Received 31 August 2006; received in revised form 8 April 2007; accepted 18 April 2007
Abstract
The young flower buds of diploid Oriental cultivars: ‘Con. Amore’ and ‘Acapulco’ (Lilium) were treated with
0.02, 0.05, 0.1, and 0.2% colchicine to induce diploid egg (female gamete). The treated young buds between the
two cultivars were crossed reciprocally as female parents with normal pollen respectively. The polyploid plants
were identified by measuring stoma size and chromosome number. The results showed: that triploid progenies
could be obtained through this way, and the fact that the treated young flower buds were successfully used as
female parents indicated the formation of 2n or 2x egg cells. The above results implied that polyploidization by
artificially induced diploid female gametes could be a powerful method to create novel variations in the breeding
of Oriental lilies. # 2007 Published by Elsevier B.V.
Keywords: Dilpoid gametes; Lilium Oriental; Triploid
1. Introduction
Lily (Lilium L., 2n = 2x = 24) is one of the most important ornamental bulb worldwide. It is
widely cultivated as a cut flower, pot and garden plant. About 1500 million bulbs are produced
each year around the world, predominantly in the Netherlands, Japan and the United States of
America, but more recently also in the Southern Hemisphere, such as, in Australia and Chile. As a
cut flower, lily is now ranked as the fourth most important crop in the Netherlands. The plant is
also cultivated widely in China as a very important cut flower.
The genus Lilium was traditionally classified into seven sections by Comber (Comber, 1947).
Revised classification (De Jong, 1974) listed three sections: Sinomartagon (the Asiatic hybrids),
Archelirion (the Oriental hybrids) and Leucolirion (the Longiflorum hybrids) as the most
economically important groups because of their wide range of flower shape, size, color and
morphological characteristics. Lilies of ornamental importance are the result of crosses within
three sections and interspecific hybrids within sections have been bred and cultivated widely since
early 1800s (Shimizu, 1987). Lilies breeders have tried to broaden the genetic basis by a number
of approaches. Among them, polyploidization has become a very effective way to increase genetic
variability, and mitotic polyploidization induced through chemical treatment (somatic
chromosome doubling) has been used in lily breeding for more than 30 years (Van Tuyl et al.,
1992). However, a major drawback of somatic chromosome doubling is the lack of intergenomic
recombination in the progenies because of strict autosyndetic pairing (Lim et al., 2003). An
alternative method is sexual polyploidization from unreduced (2n) gametes produced by
aberration in the meiotic process, which could result in intergenomic recombination in lily (Lim et
al., 2003; Barba-Gonzalez et al., 2004; Camadro and Peloquin, 1980). Although both modes can
double chromosome number, their relative efficacy is very different in genetic variability, epistasis,
heterozygosity and ability of transferring specific genetic traits (Barba-Gonzalez et al., 2004;
Beuselinck et al., 2003). It was suggested that the sexual polyploidization through 2n gametes
might produce more genetic variations than those from colchicine-induced polyploids (Beuselinck
et al., 2003). The existence of 2n-gamete will allow lily breeders to broaden the genetic basis of
cultivated hybrids. In fact, sexual polyploidization through 2n-gametes has been a major route for
naturally occurring polyploids (Harlan and De Wet, 1975; Van Tuyl, 1990). However, the selection
for the genotypes that produce 2n gametes spontaneously is a very laborious process and an
induced higher production of 2n gametes will certainly increase the breeding efficiency.
The study demonstrated that triploid hybrids could be obtained through diploid female
gametes in Oriental lilies.
2. Materials and methods
2.1. Plant material and induction of diploid gametes
Two diploid cultivars of Lilium Oriental ‘Con. Amore’ and ‘Acapulco’ were treated by 0.0,
0.02, 0.05, 0.1, and 0.2% colchicine when the young flower buds were about 0.5 cm in length and
0.5 cm in width for the production of 2n-gametes. For each variety, there are 300 young buds
treated per concentration. During the period of treatment, the treating young flower buds were
wrapped with a cotton wool soaked in different concentration of colchicine and last for three days.
All plants were grown in a glasshouse at 17–20 8C during day and at 14–18 8C during night.
Diploid pollen production was identified by microscope observation of pollen size from the
treated young buds and diploid egg cell production was judged indirectly by the
variation of stigma.
2.2. Use of diploid egg in crosses
The crosses were made: treated ‘Con. Amore’ _ normal ‘Acapulco’, treated ‘Acapulco’ _
normal ‘Con. Amore’, normal ‘Con. Amore’ _ treated ‘Acapulco’, and normal ‘Acapulco’ _
treated ‘Con. Amore’ to achieve triploid F1 progenies. For each cross, 15 young buds were
pollinated. Progeny plants from these crosses were obtained using embryo rescue method (Van
Tuyl et al., 1991). All fruits with obvious variation were collected 50–60 days after pollination,
submerged in 75% ethanol, flamed for surface sterilization and cultured in vitro to grow into
plants. Generally, swollen ovules contained an embryo and when it was possible, the embryo was
removed from the ovule and cultured in enriched media. The subculture of plantlets was done
according to the
method described by Nova´k (Nova´k and Petru´, 1981).
2.3. Chromosome preparation
Triploid plants were identified by measuring stomas size and counting the chromosome
number (3n = 3x = 36).
Root tips were used for chromosome preparations. Root tips were kept for 10 h in 0.002 mM
8-hydroxy quinoline solution at 4 8C, then fixed in Carnoy solution (ethanol: acetic acid = 3:1) for
12 h, and digested for 10 min in 0.1 M HCl. Root tips were washed for 20 min in distilled water
and squashed in 60% acetic acid. The slides were stained with 10% modified phenol-fuchsin
solution before examination under Olympus microscope.
3. Results
3.1. Effect of the colchicines treatment on the production of diploid gametes
After young flowers of ‘Con. Amore’ and ‘Acapulco’ were treated by different concentration
of colchicine, their stigma and pollen size became bigger, indicating indirectly diploid gametes
had been induced successfully. According to morphological variation—the stigma became obese
and the diploid pollen enlarged about 30% in diameter than normal haploid pollen, the
percentage of mutation was investigated (Table 1).
The sensitivity of the two cultivars to colchicines were very different. ‘Con. Amore’ was more
sensitive than ‘Acapulco’. Higher concentration of colchicines 0.2% resulted in a considerable
number of young flowers of ‘Con. Amore’ to die, so that just small part of young flowers was
induced to produce diploid gametes. The most suitable colchicines concentration of inducing
diploid gametes was same 0.1% for both cultivars, but the percentage of successfully induced
flowers for the two cultivars were rather different, i.e., 9.5% for ‘Con. Amore’ and 25.8% for
‘Acapulco’.
3.2. Crosses of treated plants with normal plants
Seven to eight weeks after pollination, the fruits were collected and cultured in vitro for about
50–60 days to grow into plants. The results of chromosome number examination showed the
ploidy levels of progenies from the cross with diploid gametes (Table 2). Although many mature
embryos and plants were obtained from the crosses between the treated pollen of ‘Con Amore’ and
normal female of ‘Acapulco’, and the treated pollen of ‘Acapulco’ and normal 1n egg of ‘Con
Amore’, no triploid progenies were observed after cytological analysis and stoma size
examination. While the crosses between the treated female of ‘Con Amore’ and normal pollen of
‘Acapulco’, and the treated female of ‘Acapulco’ and normal pollen of ‘Con Amore’ produced
triploid progenies (2n = 3x = 36) as expected.
3. 3. Mitotic behavior and chromosome number in the progenies
In the majority of vegetative descendants of triploids from treated eggs, their mitosis were
regular, but some abnormal phenomena, such as chromosome bridges, lagging chromosomes, and
multiple nuclei were observed. These abnormal phenomena during mitosis may be one of the
mechanisms of producing a great number of variations among the vegetative descendants of
triploids derived from the treated eggs.
Cytological observation of F1 hybrids produced by the induced diploid-gametes showed that
triploid plants were obtained as expected and its chromosome number is 2n = 3x = 36. However,
chromosome number variations were also observed among the vegetative descendants of these
triploid plants and some plants returned to diploid level. For the probable reasons of chromosome
variation in these unstable plants did not be studied in this paper.
4. Discussion
It is well known, there are some evident advantages of triploid lily cultivars over diploid ones,
for example (1) they have bigger leaves and higher yield, (2) they show better leaf quality and
higher resistance to a number of leaf diseases, (3) multiple copies of gene alleles accumulate in the
resulting hybrids, which are expected to show gene dosage effect and heterosis, and (4) they
normally have sterile pollens so as to avoid pollen pollution to surroundings and reduce risk to the
population sensitive to pollen (Masumi, 2003). However, the traditional method which breeds the
triploid lilies is: at first get the tetraploid by mitotic polyploidization, then crossing with normal
diploid to achieve triploid plant. Due to strict autosyndetic pairing, mitotic polyploidization is no
genetics recombination so that it’s difficult to produce genetics improvement. But it is completely
possible for the meiotic polyploidization through 2n-gametes to produce genetics recombination
and obtain genetics improvement (Lim et al., 2003; Barba-Gonzalez et al., 2004). The objective of
our research is to breed triploid lilies through 2n-gametes and evaluate the field performance of
these triploid progenies. In this study, we have demonstrated the feasibility of artificially inducing
possible 2n female gametes by colchicine and using these diploid egg cells to produce triploid
hybrids.
In comparison with traditional methods, 2n-gamete technique is able to produce triploid more
rapidly and to take the most heterotic advantages. Triploid plant could be obtained from
2ngametes producer with one simple step when crossed with the normal diploid plants.
Furthermore, only a few types of 2ngametes are produced, therefore, progeny segregations are
fewer to save the time of selection. These characteristics can speed up the breeding process and
produce new varieties quickly for the floricultural industry.
Occurrence of 2n-gametes seems to be controlled genetically, but expression of these genes is
influenced greatly by the environment (Ramanna and Jacobsen, 2003). There are reports of
different attempts to increase and even induce the production of 2n gametes. These attempts
include: genetic selection,N2O, high solar level, low temperature, heat shock and caffeine
treatments of immature flower buds (Barba-Gonzalez, 2005). This study used colchicines as
inducing agent of 2n-gametes production. The notable advantages of using colchicine compared to
other methods are: (1) its practice is very simple and short time, (2) no special equipment or
roomis required, for example it can be done in either field or greenhouse, whereas heat shock
needs a heat room (Lokker et al., 2004) and N2O treatment required a gas roomwith the pressure
instruments (Barba-Gonzalez, 2005), and (3) inducing efficiency is very high, up to 25.8%.
For combining desirable characteristics into new cultivars, interspecific hybridization is often
needed. But interspecific hybrids of distantly related species are generally sterile due to disturbing
chromosome pairing during meiosis. Although it can be overcome by doubling the somatic
chromosome number to produce fertile allotetraploid, no intergenomic recombination occurs
between the alien chromosomes during meiosis as homologous chromosomes pair preferentially in
mitotic polyploidization. Due to the intergenomic recombination between the alien genomes in
meiotic polyploidization derived from 2ngametes (Lim et al., 2003; Barba-Gonzalez et al., 2004;
Beuselinck et al., 2003), genetic variation is easily achieved to broaden the selection basis for
breeding commercial cultivars. So the meiotic polyploidization through 2n-gametes would give
lily breeders more opportunities to select new varieties.
It is evident from this research that sexual polyploidization through diploid-gametes is a
desirable and possible approach for breeding new lily varieties. The process also can introduce
more heterosis into the progeny. Wider hybridization through 2n-gametes is needed in lily
breeding for crosses within one taxonomic section have become very difficult to obtain genetic
improvement by traditional breeding techniques. In Lilium, sexual polyploidization via 2n-pollen
has been reported by many researchers (Lim et al., 2001, 2003; Barba-Gonzalez et al., 2004;
Karlov et al., 1999; Lim and Shen, 2004; Van Tuyl et al., 1989; Lim and Van Tuyl, 2004),
however the induction of 2n egg and its use is few (Zhou, 2007). Besides the first report from
N2O treatment (Barba-Gonzalez et al., 2006), this colchicine treatment could be used in lily
breeding.
Acknowledgements
The authors would like to thank the National Science and Technology Ministry (No:
05EFN215300265) for the financial support and professor Jaap M. Van Tuyl for valuable
comments to this research.
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