Table 1. Relationship between seed hair and embryo development in ((Populus pseudo simonii × P. nigra ‘Zheyin3#’) × (P. × beijingensis)) ovaries.
Defined
stages 		Developmental
characteristics
of seed hair 		Percentage of each developmental stage (%)
Developing embryo sac Mature embryo sac and double fertilization Hypnozygote 2-celled embryo 4-celled embryo 8-celled embryo >8-celled embryo
I No hair forms 83.72
(36) a) 		16.28
(7) 		         
II Development of the hair initiates around the funiculus 23.68
(9) 		63.16
(24) 		13.16
(5) 		       
III The hair gradually elongates to cover the funiculus   36.36
(16) 		56.82
(21) 		6.82
(3) 		     
IV The hair begins to enclose the ovules     44.44
(20) 		37.78 (17) 15.56 (7) 2.22
(1) 		 
V The hair encloses the ovules completely, but does not fill the ovary     9.52
(4) 		26.19 (11) 38.10 (16) 21.43 (9) 4.76
(2)
VI The hair fills the ovary         18.37 (9) 34.69 (17) 46.94 (23)
a) Number of observed ovules.
1

Fig. 1. Double fertilisation and zygote development of (P. pseudo-simonii × P. nigra ‘Zheyin3#’) × (P. × beijingensis). a-b) Serial sections showing a mature embryo sac and two sperms (arrow); c) Movement of the sperm, arrow showing a sperm is close to the egg; d) Double fertilisation, one sperm is fusing to the egg (arrow) and the other to central cell (arrow head); e) Zygote; f) Two-celled embryo; g) Four-celled embryo; h) Eight-celled embryo; i) Globular embryo. Bars are equal to 10 μm in a–h and 50 μm in i.

2

Fig. 2. Development of seed hairs in the ovaries: a) Stage I ovary without hair formation; b) Stage II ovary, indicating initiation of hairs around the funiculus; c) Stage III ovary, characterised by hair elongation gradually covering the funiculus; d) Stage IV ovary with hairs enclosing the ovules; e) Stage V ovary not filled by hairs, but completely enclosed ovules; f) Stage VI ovary filled by hairs.

3

Fig. 3. Ploidy level detection of offspring derived from zygotic chromosome doubling: a) Somatic chromosome of a tetraploid (2n = 4x = 76); b–c) Flow cytometric analyses of a diploid and tetraploid, respectively; d) Histogram of flow cytometric analysis of nuclei mixture of young leaves from the former diploid and tetraploid plants.

Table 2. Tetraploid production through colchicine-induced zygotic chromosome doubling in (P. pseudo-simonii × P. nigra ‘Zheyin3#’) × (P. × beijingensis) hybrids.
Stage of seed hair development Concentration of colchicine solution (%) Duration of treatment (h) No. of catkins No. of seeds No. of
seedlings 		No. of
tetraploids 		Efficiency of tetraploid production (%)
II 0.3 24 2 124 58 1 1.72
0.5 24 2 108 39 1 2.56
III 0.3 24 3 248 174 2 1.15
0.5 24 2 104 64 2 3.13
IV 0.3 24 2 36 6 0 0
0.5 24 2 78 28 0 0
V 0.3 24 2 85 27 0 0
0.5 24 2 79 37 0 0
Control   2 417 240 0 0
Table 3. Tetraploid production through high-temperature-induced zygotic chromosome doubling in (P. pseudo-simonii × P. nigra ‘Zheyin3#’) × (P. × beijingensis) hybrids.
Stage of seed hair development Temperature (°C) Duration of treatment (h) No. of catkins No. of seeds No. of
seedlings 		No. of
tetraploids 		Efficiency of tetraploid production (%)
II 39 2 2 132 74 0 0
4 3 172 84 0 0
6 2 153 72 1 1.39
41 2 3 217 112 0 0
4 3 232 122 1 0.82
6 4 341 295 1 0.34
43 2 2 106 32 0 0
4 2 24 - - -
6 2 17 2 0 0
III 39 2 3 208 137 3 2.19
4 3 254 142 2 1.41
6 2 131 81 2 2.47
41 2 3 219 120 0 0
4 2 137 42 1 2.38
6 2 147 44 2 4.55
43 2 2 114 26 0 0
4 2 13 - - -
6 2 - - - -
IV 39 2 2 80 21 0 0
4 2 155 92 1 1.09
6 2 177 118 3 2.54
41 2 2 149 97 2 2.06
4 2 73 23 1 4.35
6 2 74 36 0 0
43 2 2 101 27 2 7.41
4 2 24 - - -
6 2 - - - -
V 39 2 3 182 80 0 0
4 2 130 53 1 1.89
6 2 79 22 0 0
41 2 2 138 77 1 1.3
4 2 83 22 0 0
6 2 105 50 0 0
43 2 2 125 86 1 1.16
4 2 57 14 0 0
6 2 - - - -
Control   2 476 256 0  
4

Fig. 4. Stomatal size and frequency of diploid (a) and tetraploid (b) plants.

Table 4. Stomatal characteristics of diploid and tetraploid (P. pseudo-simonii × P. nigra ‘Zheyin3#’) × (P. × beijingensis) offspring.
Characters Tetraploids Diploids t-value P-value
Range Mean ± SE Range Mean ± SE
Stomata length (μm) 31.9–42.6 37.6 ± 0.4 24.3–33.2 28.9 ± 0.5 14.350** <0.0001
Stomata width (μm) 18.0–23.5 20.4 ± 0.2 15.0–19.8 17.1 ± 0.2 10.178** <0.0001
Stomatal frequency (mm–2) 57.7–180.6 113.6 ± 5.4 146.7–294.3 219.6 ± 7.7 11.142** <0.0001
** Significant differences between diploids and tetraploids at α = 0.01 in t-test.