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Induction of Diploid Eggs With Colchicine During
Embryo Sac Development in Populus
By J. WANG1),2), X. Y. KANG1),2),*), D. L. LI2), H. W. CHEN2) and P. D. ZHANG1),2) (Received 22nd August 2009) induction of 2n pollen, due to easy screening by their Diploid (2n) eggs were induced by treating developing size. JOHNSSON and EKLUNDH (1940) first induced 2n embryo sacs of Populus with colchicine solution, in order pollen of P. tremula and P. tremuloides with colchicine, to produce triploid plants. The optimal pollinated time and produced triploid plants by pollinating to female of female catkins was confirmed as timing point for each catkins of diploid plants with the artificial 2n pollen. So treatment. When female catkins of P. pseudo-simonii x far, more triploids have been obtained by crossing with P. nigra ‘Zheyin3#' had become 5.62 ± 0.13 cm long 84 h induced 2n pollen in P. deltoides, P. alba, P. tomentosa after they emerged from their bract scales and all stig- and other white poplars (MASHKINA et al., 1989; ZHANG mas were exposed, pistils all over the entire catkin had and LI, 1992; KANG et al., 2000), and the pachytene optimal stigma receptivity. Observation of paraffin sec- stage of microsporogenesis is proved as the optimal tions showed that embryo sac development of ‘Zheyin3#',which initiated 12 h before pollination and finished stage for 2n pollen induction with colchicine. Although 132 h after pollination, was a successive and asynchro- the percentage of artificial 2n pollen can be upward of nous process. Generative cell division of pollen of the 80 % (ZHANG and LI, 1992; KANG et al., 1999), the effect male parent P. x beijingensis took place 3–16 h after pol- of triploid production is not good because of competition lination. Catkins of 18–96 h after pollination of from normal pollen (KANG and ZHU, 1997; KANG, 2002).
‘Zheyin3#' were treated with colchicine solution. In the Compared with 2n pollen, triploid production via 2n progeny, twenty three triploids were detected by chro- eggs may be more suitable. However, reports on induc- mosome counting and the highest rate of triploids was tion of 2n eggs are rare in Populus, because it is difficult 66.7 % in one treatment. The rate of triploid yield waspositively correlated with the frequency of four-nucleate to timely determine the exact stage of megasporogene- embryo sacs (r = 0.6721, p = 0.0981) and was not sig- sis, which progresses in the inside of ovules. Based on nificantly correlated with the percentages of uni-, two- the temporal relationship between megasporogenesis and eight-nucleate embryo sac (r = –0.1667, p = 0.7210, and microspore development under similar cultured con- r = –0.3069, p = 0.5031 and r = 0.0189, p = 0.9679, ditions in P. alba x P. glandulosa, LI et al. (2008) respectively), suggesting that the third mitotic division obtained 12 triploids by inducing unreduced megaspores of embryo sac may be the effective stage to induce 2n with a 0.5 % colchicine solution at the prophase of the eggs. Through this approach, completely homozygous first meiotic division, and the highest rate of produced 2n eggs can be produced. Its significance for plant breed- triploids in one treatment was 16.7 %. It is well known ing is discussed.
that functional megaspore develops into mature female Key words: 2n egg, colchicine, embryo sac development, Popu- gametophyte after megasporogenesis, i.e. megagameto- genesis (also called embryo sac development). In thisprocess, the megaspore undergoes at least one round of mitosis without cytokinesis. For Populus, the embryosac development complies with Polygonum type, in The genus Populus, as an important source of fuel, which a 7-celled mature embryo sac formation initiated fibre and lumber, is widely distributed and cultivated from a functional megaspore via three rounds of mitotic over the northern hemisphere (RAE et al., 2007). Some division (NAGARAJ, 1952; KIMURA, 1955, 1963; LI et al., triploid cultivars of Populus have many desirable prop- 1982; LI and ZHU, 1988; ZHU and LI, 1989; DONG, 1984; erties in growth and pulpwood characteristics compared FAN, 1984; LI and MA, 2006). Potentially, these mitotic to diploids (VAN BUIJTENEN et al., 1958; EINSPAHR et al., divisions offer us the possibility to induce 2n eggs.
1972; ZHU et al., 1995). Thus, triploid breeding is one ofthe most powerful approaches for improvement of Popu- Colchicine is an alkaloid that contributes to the pre- vention of tubulin polymerization, thereby arresting for-mation of spindle and restraining nuclear division at Diploid (2n) gametes are usually applied to produce metaphase (JORDAN and WILSON, 1999). Consequently, triploids of Populus. There are many investigations on colchicine has been widely used to induce polyploids inplants (EIGSTI and DUSTIN, 1955). In polyploid breeding 1) National Engineering Laboratory for Tree Breeding, Beijing programs of the genus Populus, colchicine is the most Forestry University, 100083, Beijing, P. R. China.
commonly used reagent and has good effects (JOHNSSON 2) Key Laboratory of Genetics and Breeding in Forest Trees and and EKLUNDH, 1940; EINSPAHR, 1965; KANG et al., 1999, Ornamental Plants, Ministry of Education, Beijing Forestry 2004; LI et al., 2008). In our investigation, female University, 100083, Beijing, P.R. China.
catkins of P. pseudo-simonii x P. nigra ‘Zheyin3#' under *) Address correspondence to: P.O. Box 118, Beijing Forestry embryo sac development were treated with colchicine University, 100083, Beijing, P.R. China; Tel.: +86-10-62336104;E-Mail: [email protected] solution, in order to discuss the possibility of 2n egg Silvae Genetica 59, 1 (2010)

induction and to serve to triploid breeding programs of The branches were cultured in a greenhouse (10–20 °C) the genus Populus.
Determination of optimal stigma receptivity Materials and Methods
Female catkins were selected to examine stigma Plant materials receptivity of ‘Zheyin3#' every 6 h, starting with the Female floral branches of P. pseudo-simonii x P. nigra catkins emerging from bract scales. Lengths of the ‘Zheyin3#' (female parent, 2n = 2x = 38) were collected catkins and colour of their stigmas were recorded. For from a plantation in Tongliao City (Inner Mongolia each time, the lengths of 10 catkins were measured with Autonomous Region, P. R. China). Male floral branches a vernier caliper. The catkins were hand-pollinated with of P. x beijingensis (male parent, 2n = 2x = 38) were col- fresh pollen of P. x beijingensis. Four hours after pollina- lected from the campus of Beijing Forestry University.
tion, the catkins were fixed in FAA (70 % ethanol: acetic Figure 1. – Stigma receptivity of P. pseudo-simonii x P. nigra ‘Zheyin3#'. Lower-case let-ters a, b and c show pollen germination on stigmas at base, middle and top of catkinsrespectively. Unmarked bars are equal to 100 µm.
Fig. 1: Catkin before optimal pollinated time. Normal pollen germination on basal andmiddle stigmas (1a and 1b). Only a few pollen grains germinated on the top stigmas(1c).
Fig. 2: Catkin at optimal pollinated time. Vigorous pollen germination on stigmas at dif-ferent parts of the catkin.
Fig. 3: Catkin after optimal pollinated time. Pollen germination on the basal stigmas(3a) is not as good as that on the middle and top stigmas (3b and 3c).
Fig. 4: Catkin with retained receptivity. No receptivity on the basal stigmas (4a), tardygermination on the middle stigmas (4b) and relatively normal germination on the topstigmas (4c).

acid: 40 % formaldehyde, 90:5:5) for 24 h at 4 °C. After opment of generative cells was examined under the being washed with distilled water, the stigmas at the above fluorescence microscope.
top, middle and base of each fixed catkin were respec-tively softened in 8 M NaOH for 2 h. Following further Determination of developmental process of embryo sacs washing in distilled water, the flowers were squashed in The female buds and catkins of ‘Zheyin3#' were fixed 0.1% aniline blue and stained for 10 min. The prepara- in FAA at 4 °C every 12 h after being cultured and every tions were observed under a fluorescence microscope 6 h after pollination, until maturation of seeds. Ovaries (Olympus BX51).
from each fixed buds and catkin were embedded withparaffin and sectioned at 8–10 µm. The sections were Observation of generative cell division in pollen stained with iron-hematoxylin and observed under the The development of generative cells was studied in microscope. During the embryo sac development, a total vivo. Pollen grains of P. x beijingensis were pollinated to of 1,470 sacs were analyzed in order to reveal the stigmas with optimal receptivity of the female parent.
process of embryo sac development.
The stigmas were fixed in FAA every 2 h for 24 h at 4 °C,starting from being pollinated until 48 h after pollina- Induction of 2n eggs tion. Subsequently, they were softened in 8 M NaOH for When female catkins acquired the optimal receptivity, at least 12 h. After being washed in distilled water, the they were pollinated with fresh collected pollen of samples were squashed in one drop of 4',6-diamidino-2- P. x beijingensis. Based on the process of the embryo sac phenylindole (DAPI) and stained for 10 min. The devel- development of ‘Zheyin3#', the female catkins 18–96 h Figure 5. – Generative cell division of P. x beijingensis in vivo. Two-celled pollen (a), mitoticmetaphase (b), anaphase (c) and two formed sperms in tube (d). Bars are equal to 10 µm.
Figure 6. – Embryo sac development and fertilization of P. pseudo-simonii xP. nigra ‘Zheyin3#'. The chalazal end is at the top in all figures. a, Uni-nucleateembryo sac with three degenerated megaspores. b, Two-nucleate embryo sac. c,Four-nucleate embryo sac. d–e, Mature embryo sac including three antipodalcells (d), one secondary nucleus and one egg apparatus (e). One of the synergidcells is out of focus. f, Fertilization. The arrow shows one sperm is close to egg.
Bars are equal to 10 µm.
after pollination were immersed in 0.3–0.5 % colchicine during 18 h to 42 h after pollination. After treatment, solution for 18–30 h. In view of the weak colchicine-tol- the colchicine was rinsed away under taps. The catkins erance of catkins soon after pollination, both 0.3 % and without treatment were set as the control group. After 0.5 % colchicine solution were used to treat the catkins the catkins matured, seeds were collected and germinat- Table 1. – Process of embryo sac development in P. pseudo-simonii x P. nigra ‘Zheyin3#'.

ed in soil. When the seedlings grew to about 30 cm in 4a). The middle and top stigmas were still green-yellow.
height, they were transplanted to the field.
Pollen germination on the middle and top stigmas wasbetter than on the basal stigmas, but not flourishing Analysis of ploidy level (Figs. 4b and c). Seven days after they emerged from The determination of the ploidy level was conducted their bract scales, all stigmas at the catkins became by somatic chromosome counting. Stem tips were brown and pollen no longer germinated.
removed from the seedlings and pretreated with a satu-rated solution of paradichlorobenzene for 4 h at 25 °C.
Division of generative cells in pollen of P. x beijingensis Subsequently, the materials were fixed in a fresh Mature pollen grains of P. x beijingensis were of the 2- Carnoy solution (ethanol: acetic acid, 3:1) for 24 h at celled type (Fig. 5a). In vivo, pollen grains started to 4 °C. The fixed materials were then hydrolyzed in a germinate on the stigmas of ‘Zheyin3#' 2 h after pollina- mixed fluid of 38 % HCl and ethanol (1:1) for 25 min at tion. Generative cell division occurred 3–16 h after polli- room temperature. After being washed in distilled water nation (Figs. 5b and c). Two sperm cells were formed three times for 15 min, the hydrolyzed materials were 12–18 h after pollination (Fig. 5d).
squashed heavily in a Carbol Fuchsin solution. Chromo-some counting was carried out under the Olympus Development of embryo sacs BX51. At least 20 cells with a well-spread metaphase Embryo sac development of ‘Zheyin3#' was of the typi- from each seedling were observed.
cal Polygonum type. Functional megaspore formed a 7-celled mature embryo sac via three rounds of mitotic divisions (Fig. 6). The mature embryo sac consisted oftwo synergid cells, an egg cell, a central cell and three Stigma receptivity of ‘Zheyin3#' antipodal cells (Figs. 6d and e). The stigma receptivity of ‘Zheyin3#' lasted 3–4 d. The The embryo sac development was a successive and receptivity among pistils in different parts of catkin var- asynchronous process (Table 1). It was initiated 12 h ied. The pistils at the base of the catkin were the first to before pollination. Three micropylar megaspores of a acquire stigma receptivity and were also the first to lose tetrad began to degenerate and the enlarged functional it; the pistils at the top of the catkin acquired it at last megaspore at the chalazal end formed a uni-nucleate but were also the last to lose it.
embryo sac (Fig. 6a). Until 24 h after pollination, the Sixty hours after the catkins emerged from their bract uni-nucleate embryo sac was dominant, although other scales, they developed into 3.45 ± 0.06 cm long. All stig- stages, such as the tetrad, the two-nucleate embryo sac mas in the catkins were green-yellow in colour. Bracts of (Fig. 6b), the four-nucleate sac (Fig. 6c) and even the basal pistils evaginated and stigmas were exposed. After eight-nucleate sac (Figs. 6d and e), were also observed.
pollination, pollen germination was observed on the Thirty to forty-eight hours after pollination, the propor- basal stigmas. However, stigmas at the middle and top tion of the two-nucleate embryo sac was predominant.
of the catkins, covered under bracts, had not acquired With the development of embryo sacs, the rate of the receptivity. Twelve hours later, the catkins were four-nucleate embryo sac increased gradually. Its per- 4.23 ± 0.13 cm in length. Except for the top stigmas, the centage was greater than that of other stages 54–60 h middle and basal stigmas had both emerged from their after pollination. After that, all cells developed into bracts (Fig. 1). Pollen germination tests showed that the eight-nucleate and mature embryo sacs in succession.
stigmas at the middle and base of the catkins both had Fertilization occurred between 84 h and 144 h after pol- receptivity (Figs. 1a and b). Only a few pollen grains lination (Fig. 6f).
with little germination adhered to the top stigmas(Fig. 1c). With their development, the catkins elongated Production of triploids to 5.62 ± 0.13 cm long 84 h after they emerged from their Table 2 shows that a total of 23 triploids (2n = 3x = 57, bract scales. All bracts turned inside out, some even Fig. 7a) were obtained by treating pollinated catkins of were shed. At this stage, all stigmas were exposed ‘Zheyin3#', under embryo sac development. In some (Fig. 2). Pollen germination tests showed that plenty ofpollen germinated vigorously on all stigmas at differentparts of the catkins (Figs. 2a, b and c), which indicatedthat the catkins acquired the optimal stigma receptivity.
Therefore, this time was the optimal pollinated time.
When the catkins developed to 6.27 ± 0.10 cm in length102 h after they emerged from their bract scales, theybegan to become less incompact (Fig. 3). Although pollengrains still germinated on all stigmas, the number ofgerminated pollen grains on the basal stigmas was lessthan that on the middle and top stigmas (Figs. 3a, band c). Five to six days after they emerged from theirbract scales, the catkins relaxed completely and were10.26 ± 0.18 cm long (Fig. 4). The basal stigmas became Figure 7. – Number of Chromosomes of diploid and triploid yellow-green in colour with a little brown. Only a few progenies. Fifty seven chromosomes in triploid (a) and thirty pollen grains clung to the stigmas and germinated (Fig. eight in diploid (b). Bar is equal to 10 µm.
Table 2. – Triploid production through treating developing embryo sacs of P. pseudo-simonii x P. nigra‘Zheyin3#' with colchicine solution.
treatments, no seedlings survived. All seedlings in the yield in progeny. Therefore, exact pollination should be control group were confirmed as diploid (2n = 2x = 38, emphasized. In the present study, the optimal stigma Fig. 7b). Nineteen triploids were produced in the treat- receptivity of catkin of ‘Zheyin3#' was identified, which ments 54–66 h after pollination, representing 82.6 % of insured successful hybridization and reliable seed set.
the total number of triploids. The highest rate of Furthermore, treatment of 2n egg induction is based on triploids in one treatment was 66.7 %, which occurred 60 timely determination of stage of embryo sac develop- h after pollination.
ment, but it is difficult to detect because of its location Correlation analyses were made between the efficien- inside of ovule. In view of the result that the embryo sac cy of triploid production and percentage of each develop- development of ‘Zheyin3#' almost undertook after polli- mental stage of embryo sac (Fig. 8). A moderate positive nation, the optimal pollinated time was useful to be correlation was found between the rate of triploids and regarded as a reference point for each treatment in this the percentage of four-nucleate embryo sacs (r = 0.6721, p = 0.0981). However, there were no significant corre- There are two types of mature pollen in plants, i.e. 2- lations between the rate of triploids and the percent- celled and 3-celled types. For 2-celled pollen, division of ages of uni-, two- and eight-nucleate embryo sacs generative cell occurs in pollen tube after germination.
(r = –0.1667, p = 0.7210, r = -0.3069, p = 0.5031 and EINSPAHR (1965) unexpectedly obtained some putative r = 0.0189, p = 0.9679, respectively).
triploids when he treated catkins in 6–30 h after polli-nation in order to induce tetraploids by treating newlyformed embryos of quaking aspen with colchicine. WIN- TON (1968) explained that production of these triploids Pollination at the right time is the basis for successful was caused by an unreduced division of generative cell hybridization. Stigma receptivity is an important factor in pollen tube. In our investigation, however, the gener- affecting effective pollination, which is related to seed ative cell of pollen of male parent P. x beijingensis divid- yield (EGEA et al., 1991; SANZOL and HERRERO, 2001).
ed 3–16 h after pollination, which did not coincide with Estimation of 2n egg production relies on the polyploid the effective period of triploid production. It suggests Figure 8. – Correlation analyses between rate of triploids and percentage of each embryo sac developmental stage.
that the triploids in this study are not from fertilization catkins under embryo sac development and the highest of diploid generative cell. rate was up to 66.7 % in one treatment, which suggests In the genus Populus, induction of 2n gametes has, so that a novel approach for 2n egg induction has been dis- far, focused on the meiotic process. Prophase I is an effi- covered and that this approach is more efficient for cient stage to induce 2n gametes. For pollen chromo- triploid production than both the 2n pollen and 2n some doubling, the percentage of 2n pollen can be megaspore approaches. Further more, correlation analy- upward of 80% by treating with colchicine (Z ses showed that the rate of triploid production was posi- tively correlated with the frequency of four-nucleate I, 1992; KANG et al., 1999). However, it is not compati- ble between the high percentage of 2n pollen and the embryo sac, but not significantly correlated with both rate of triploid production by pollinating with them, uni- and two-nucleate sacs, indicating that the third owing to the weak competitive ability of 2n pollen com- mitotic division during embryo sac development may be pared with normal pollen in germination and fertiliza- more effective for 2n egg induction through external actions, but it can not exclude the possibility of 2n egg ANG and ZHU, 1997; KANG, 2002). The highest rate of triploid production by pollinating with 2n pollen formation by mitotic inhibition at the first or second was just 12.9%, even though the 2n pollen had been treated by γ-irradiation to enhance their competitive KANG et al. (2004) obtained 57.1% allotriploids of ability (KANG et al., 2000). For female gamete chromo- white poplar by treating female catkins with colchicine some doubling, the effect just can be estimated by poly- 24–36 h after pollination. However, he did not give a ploidy production in progeny. LI et al. (2008) obtained 12 reasonable explanation on origin of these triploids. Addi- triploids by crossing with 2n megaspores induced by tionally, when EINSPAHR (1965) treated pollinated colchicine at prophase I of megasporogenesis and the catkins of quaking aspen to induce tetraploid, he unex- highest rate of triploid production was 16.7 % in one pectedly screened some putative triploids. Although treatment. In the present investigation, twenty-three chromosome doubling of sperm cells is possible (WINTON, triploids were produced by treating pollinated female 1968), production of 2n eggs during embryo sac develop- ment may be a better explanation for the results of the earlier studies, in view of its high efficiency.
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Herausgeber: Johann Heinrich von Thünen-Institut. Bundesforschungsinstitut für Ländliche Räume, Wald und Fischerei.
Schriftleitung: Institut für Forstgenetik, Sieker Landstraße 2, D-22927 Großhansdorf Verlag: J. D. Sauerländer's Verlag, Finkenhofstraße 21, D-60322 Frankfurt a. M.
Anzeigenverwaltung: J. D. Sauerländer's Verlag, Frankfurt am Main.
Satz und Druck: ADN Offsetdruck, Battenberg — Printed in Germany.
J. D. Sauerländer's Verlag, Frankfurt a. M., 2010

Source: http://germanjournalofforestresearch.de/fileadmin/content/dokument/archiv/silvaegenetica/59_2010/Heft1/_05__Wang_2364.pdf