ausgabe 55/2

Triploid Induction in Populus alba x P. glandulosa by Chromosome
Doubling of Female Gametes
By Y. H. LI1), X. Y. KANG1),*), S. D. WANG1), Z. H. ZHANG1) and H. W. CHEN1) Abstract
triploids. Triploids were obtained by chromosome dou- Efficient methods for obtaining unreduced female bling of female gametes from Morus alba L. (DWIVEDI et gametes are needed for triploid induction in Populus al., 1989) and P. tomentosa x P. bolleana (LI et al., 2001).
alba x P. glandulosa. A cytological study of MMCs and The incidence of triploid poplars, however, was low and PMCs meiosis and staminate floral elongation revealed consistent results were difficult to obtain in repeated that there was a close association between MMCs meiot- experiments. These studies identified the need to corre- ic stage and male bud phenology during development late meiotic stage of megaspore mother cells (MMCs) under the same environment conditions. Female buds of with colchicine treatment. Unfortunately, MMCs meio- Populus alba x P. glandulosa were treated with 0.5 % sis can only be observed through paraffin sectioning, colchicine solution at the selected meiotic stages based which is a slow and destructive process. This study was on male bud phenology. The treated buds were pollinat- conducted to examine the feasibility of an alternative, ed with pollen of P. tomentosa. A high rate of unreducedfemale gametes occurred in late leptotene and non-destructive approach to induce unreduced female pachytene stages of MMC, as evidenced by a relatively gametes in P. alba x P. glandulosa. We will attempt to higher percentage of triploids in the progeny, when com- correlate meiosis in pollen mother cells (PMCs) with pared to progeny from colchincine application at other male bud phenological development and in turn corre- meiotic stages. The present results demonstrated that late male bud phenology with MMCs meiosis. We will male bud phenology can be used to guide colchicine then study the effects of colchicine treatments at differ- applications to female buds and cause a more efficient ent MMCs meiotic stages on ploidy, as indicated per- Key words: chromosome doubling, effective treatment stage,female gamete, Populus alba x P. glandulosa, triploid.
Material and Methods
Introduction
In the present study, cuttings from sexually mature Triploid plants are often characterized by properties of trees of P. alba x P. glandulosa, which is native to Korea, fast growth, large leaves and vigor in comparison with and P. tomentosa, a timber species native to China, their diploid counterparts. Natural or induced triploid were. In early January 2004, floral branches were cut aspen and poplars (Populus L.) can have certain growth and hydroponically cultured in a greenhouse with day and fiber characteristics that are superior to diploid and night temperatures around 20 °C and 10 °C, respec- forms (cf. SCHLARBAUM, 1991). The heterosis of triploids in forest trees is usually the result of an increase inploidy level from fertilization between n gametes with unreduced (2n) gametes (ZHU et al., 1995). In tree breed- The morphological stage of male and female catkins ing, triploids have been induced by chromosome dou- were observed after floral branches were cultured in the bling of pollen in Populus tremula L., P. tremuloides greenhouse. The ratio of unfolded catkin/bud were mea- Michx., P. deltoides Bartr. (JOHNSSON and EKLUNDH, sured every 12 h, from 5 to 8 days in greenhouse condi- 1940; WINTON, 1968), P. alba L. (MASHKINA et al., 1989), Hevea brasiliensis Mull. (ZHENG et al., 1983), P. tomen-tosa Carr. x P. bolleana Lauche P. alba x P. glandulosa (ZHANG and LI, 1992; KANG et al., 1999). Observations of MMCs and PMCs meiosis were peri- KANG and ZHU (1997) found that 2n pollen germinated odically conducted after the female and male floral later on stigma and pollen tube grew slower than that of branches of P. alba x P. glandulosa were cut and cul- n pollen in P. tomentosa Carr. and P. tomentosa x P. bol- tured in greenhouse. After 14 h in culture, 3–4 floral leana, which suggested that 2n pollen does not have buds were collected from the female and male floral competitive advantage over n pollen and therefore, has branches every 3 h until the ninth day. The number of less chance of participating in fertilization. In contrast, floral buds collected was about 702. The collected female fertilization of 2n female gametes by n pollen would and male floral buds were fixed in FAA (18:1:1, ethy1 have the potential to create greater numbers of alcohol: formaldehyde: acetic acid) and Carnoy (3:1ethy1 alcohol: acetic acid) respectively, then stored at 1) Key Laboratory for Genetics and Breeding of Forest Trees and 4 °C. Meiosis was observed in PMCs excised from Ornamental Plants, Ministry of Education, Beijing Forestry anthers of the fixed floral buds. The PMCs were University, P. O. Box 118, 100083, Beijing, China.
squeezed out of the anthers into a drop of aceto-carmine ) Corresponding author: XIANGYANG KANG. Telephone: 86-10- 62336104, E-mail: [email protected].
Pistillate flowers at various developmental stages were removed from catkins, dehydrated by consecutive The ratio of triploids/seedlings was calculated in each immersion in 70 %, 85 %, 95 % and 100 % ethanol (2 h for colchicine treatment. The meiotic stage of MMCs was each immersion), embedded in paraffin (MA et al, 2002) determined by the percentage of MMCs meiotic stage in sectioned between 8–10 µm, and dyed with 0.5 % hema- each floral development. Pearson’s correlation coefficient toxylin. The preparations were observed under an Olym- was used to evaluate correlation between the ratio of pus BX51 microscope and photographed. At least 3–4 triploids/seedlings and the percentage of MMCs meiotic buds were sampled and 50–60 ovules were checked for stage. Statistical analysis were performed by one-way analysis of correlation coefficient using Data processingsystem 7.55 software(DPS7.55). Statiscial significant Chromosome doubling of female gametes difference was assumed when P value was < 0.05. After associating MMCs and PMCs meiotic stages with male bud development, eight female floral buds of P. alba x P. glandulosa were immersed in a 0.5 % solu- Association of MMCs and PMCs of P. alba x P. glandu- tion of colchicine (LI et al., 2001) for 24 h with the excep- tion of the controls (four female floral buds). Colchicinetreatments were initiated at 140 h, 152 h, 164 h, 176 h Cytological observations of MMCs and PMCs meiosis and 188 h respectively after the floral branches were revealed that there was a close association betweenMMCs meiotic stage and male bud phenology during cultured in greenhouse ranging from tetrad to two celled development (Table 1). After 140 h in culture, when the pollen stage of PMCs during each meiotic stage of meiosis stage of PMCs developed into tetrads, the meio- MMCs (leptotene, late leptotene, pachytene, diplotene, sis stage of MMCs of P. alba x P. glandulosa was just diakinesis and metaphase I). Pollination was conducted beginning leptotene, and 1/4 pistillate catkin and 1/2 with P. tomentosa when the stigma was at the optimal staminate catkin were protruded from squama respec- receptive stage, as evidenced by a large amount of secre- tively. After 164 h in culture, the meiosis stage of MMCs tion After pollination, the female floral branches were was in pachytene stage, and 3/4 staminate catkin and further cultured in water until seeds matured and were 1/2 pistillate catkin were protruded from squama harvested, approximately 2–3 weeks. Subsequently the respectively. Within the female buds in a pistillate seeds were sown in soil for each treatment and control.
catkin, most MMCs were synchronized meiotically.
After 2 months the seedlings were transplanted to field.
Table1 also showed the association between the morpho-logical characteristics of catkins and the stage of Chromosome counts of two-year-old hybrid progenies were determined using shoot tips prepared according to the protocol developed by KANG (1996) and stained with Table 2 presents the results obtained in terms of num- ber of seedlings and percentage of triploid poplars Table 1. – Association of MMCs and PMCs of P. alba x P. glandulosa. a Hours after culture indicates the collection time of female buds.
b unfolded catkin/bud indicates the ratio of unfolded catkin/bud.
Figure 1. – Chromosomes of triploid poplars. (2n = 3x = 57).
Figure 2. – Chromosomes of diploid hybrid progeny. (2n = 2x = 38).
among the hybrid progenies. A total of 12 seedlings were percentage of triploids were obtained when the meiosis identified as triploid poplars among 354 colchicine treat- stage of most MMCs developed into late leptotene and ment hybrid progenies (Figure 1), while the remaining seedlings were diploid (Figure 2). Triploids were Statistical analysis indicated that the rate of triploids obtained in every colchicine treatment. There were no was positive correlated with the late leptotene and triploids obtained in control treatment. Guided by the pachytene stage of MMCs meiotic stage, especially in association between MMCs meiotic stage and male bud diplotene stage, while the rate of triploids had negative phenology during development, the female buds were correlation coefficient with the leptotene, diakinesis and treated with colchicine solution at the meiotic stage of metaphase I stage of MMCs meiotic stage. There was late leptotene (100 % MMCs). Two triploids were identi- not statistically significant correlation between the rate fied, accounting for 16.7% of the 12 survived two-year- of triploids and percentage of MMCs meiotic stage old seedlings. When treatment occurred at pachytene (61.5 % MMCs) two triploid poplars were producedamong 87 seedlings total. Later pachytene treatments(75 % MMCs) produced three triploid poplars among 25 Discussion
seedlings total. Treatment at diakinesis (64.7 % MMCs) The high rate of triploids was primarily obtained in produced four triploids, which represented 5.0 % of 80 MMC late leptotene and pachytene stages and were hybrid progenies. Overall, Table 2 shows that the high associated with PMC and male bud phenology develop- Table 2. – Triploid induction in P. alba x P. glandulosa by the chromosome doubling of female gamete.
c Hours after culture indicates the collection time of female buds and the treatment time of female buds.
d Control: no colchicine treatment. ment of P. alba x P. glandulosa. Although not statistical- KANG, X. Y. and Z. T. ZHU (1997): A study on the 2n pollen ly significant, the correlation trends suggests that a rel- vitality and germinant characteristics of white poplars.
atively higher frequency of unreduced 2n female Acta Botanica Yunnanica 19 (4): 402–406.
gametes were induced in late leptotene and pachytene.
KANG, X. Y. (1996): Chromosome count and shape of It may be the induction of triploidy is so low that the poplar. Journal of Gansu Agricultural University 31(1):
67–70.
statistical procedures used in this study would not show KANG, X. Y., Z. T. ZHU and H. B. LIN (1999): Study on the significance at the common error level of 0.05. Future effective treating period for pollen chromosome dou- studies that generate large number of seeds are needed bling of Populus tomentosa x P. bolleana. Scientia Silvae Sinicae 35(4): 21–24.
The high percentage of triploids (16.7 %) obtained in KANG, X. Y., Z. T. ZHU and H. B. LIN (2000): Radiosensitiv- the present study was higher than that of triploids ity of different ploidy pollen in poplar and its applica- (3.8 %) induced by the unreduced pollen in Populus tion. Acta Genetica Sinica 27(1): 78–82.
ANG et al., 2000). Therefore, colchicine induction of 2n I, Y., Z. T. ZHU, Y. T. TIAN, Z. Y. ZHANG and X. Y. KANG female gametes using the association of MMCs with (2001): Studies on obtaining triploids by colchicinetreating female flower buds of white poplars. Scientia male bud phenology as a guide appears to be an efficient Silvae Sinicae 37(5): 68–74.
approach for creation of triploids in P. alba x P. glandu- MASHKINA, O. S., L. M. BURDAEVA, M. M. BELOZEROVA and losa. Moreover, this approach avoids subjection of L. N. V’YUNOVA (1989): A method of inducing diploid female floral buds to high temperatures and daily appli- pollen in woody species. Lesovedenie 1: 19–25 (in Russ-
cations of colchicine used in previous studies (LI et al, MA, S.-M., X.-L. YE, N.-X. ZHAO and C.-Y. LIANG (2002): Embryological studies on facultative apomixis in apluda
mutica (Gramineae). Acta Botanica Sinica 44(3):
Acknowledgements
SCHLARBAUM, S. E. (1991): Cytogenetics of forest tree The authors thank Dr. G. HAZENBERG for critical read- species. In: TSUCHIYA, T. and P. K. GUPTA, eds. Chromo- ing of the manuscript. The research was financially sup- some engineering in plant genetics and breeding. Vol.
ported by the State Forestry Administration of China Elsevier Science Publ., Netherlands, p. 593–618.
(1999-12) and the National Natural Science Foundation of WINTON, L. L. (1968): Fertilization in forced quaking aspen and cottonwood. Silvae Genetica 17(1): 20–21.
ZHANG, Z. Y. and F. L. LI (1992): Studies on chromosome doubling and triploid breeding of white poplar (_)-The References
techniques of the pollen chromosome doubling, Journal
of Beijing Forestry University, vol. 14, Supp. 3: 52–5.
DWIVEDI, N. K., N. SURYANARAYANA, A. K. SIKDAR, B. N.
ZHENG, X. Q., X. S. ZENG, X. M. CHEN and G. L. YANG SUSHEELAMMA and M. S. JOLLY (1989): Cytomorphologi- (1983): A new method for inducing triploid of Hevea cal studies in triploid mulberry evolved by diploidiza- brasiliensis. Chinese Journal of Tropical Crops 4(1):
tion of female gamete cells. Cytologia 54: 13–19.
JOHNSSON, H. and C. EKLUNDH (1940): Colchicine treat- ZHU, Z. T., H. B. LIN and X. Y. KANG (1995): Studies on all ment as a method in breeding hardwood species.
triploid breeding of Populus tomentosa B301 clones.
Svensk Papp Tidn 43: 373–377.
Scientia Silvae Sinicae 31(6): 499–505.

Source: http://www.allgemeineforstundjagdzeitung.de/fileadmin/content/dokument/archiv/silvaegenetica/57_2008/Heft_1/57-1-37.pdf