Somatic Embryogenesis In Rosa Chinensis CV. ‘Parson’s Pink China’

This study investigated the effects of explant type, plant growth regulator concentration, 15 callis status, medium conversion time, and medium tilt on the growth of rose somatic embryos. 16 The results showed that Rosa chinensis cv. ‘Parson’s Pink China’ leaves could induce normal 17 embryogenic calli but petioles could not. When the 2,4-dichlorophenoxyacetic acid 18 concentration was 3.0 mg/L, the callis induction rate was the highest in the embryo 19 proliferation medium (EP medium) supplemented with 0.5mg/L kinetin, and white and 20 reddish-brown translucent calli were the main type of embryogenic calli induced. As the culture 21 time in EP medium was extended, the relative induction rate for secondary embryos and 22 multicotyledon secondary embryos gradually increased when transferred to embryo 23 maturation medium (EM medium), but the induction rate for somatic embryos decreased. 24 Placing the EM medium at an angle of 45° made the somatic embryos germinate faster and 25 the germination rate was also higher. The germination buds produced by the somatic embryos 26 with two cotyledons showed the fastest germination and greatest survival rates. The results of this experiment will help improve somatic embryo regeneration rates and explore


38
Rose is a traditional Chinese flower that has great application value. In addition to being 39 used as fresh cut flowers, it also plays an important role in the food and cosmetic industries.      intensity was about 27 μmol/m 2 /s, the culture temperature was 25 ± 2°C, and the medium was 175 changed every 4 weeks. The somatic embryos were cultured in EM medium to the 20th week.

176
The germination rate for the somatic embryos was measured and then they were transferred 177 to shoot proliferation medium (SP medium) for plant regeneration. The SP medium contained

195
The percentage data were arcsine transformed prior to ANOVA to stabilize the variance.

196
Related data were analyzed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA) and compared 197 using least significant difference tests at the 5 % probability level.  Table 1 shows that during the dark culture of EP medium for 6 weeks, the average calli 203 8 induction rates for the leaves (68.89%) were higher than those for the petioles (24.44%),

204
indicating that EP medium is more suitable for the induction of leaf calli. When the 2,4-D 205 concentration was 3 mg/L, the induction rate for leaf calli was the highest at 100%. During the 206 culture process, the leaves and petioles all produced four types of calli. These were white 207 sandy calli, white ( Fig. 1A-a) and reddish-brown ( Fig. 1A-b)

227
Embryogenic calli cultured in EP medium for 17 weeks induced three kinds of somatic 228 embryos with secondary embryos during the 9th week in EM medium ( Fig. 2A-C), which were 229 cultured in EP medium to the 21th week. This induced the production of multicotyledonous 230 embryos with secondary embryos (Fig. 2D). These secondary embryos were located around 231 the base of the somatic embryos ( Fig. 2A-D). The different somatic embryos were counted 9 after they had been cultured in EM medium to the 11th week. The results showed that, 233 extending the culture time in EP medium led to a gradual increase in the relative induction rate 234 of secondary embryos and multicotyledon secondary embryos when transferred to EM 235 medium, but the induction rate of somatic embryos decreased. This shows that after 236 embryogenic calli are formed, the medium needs to be changed to reduce the concentration of 237 cytokinin so that somatic embryos can be induced normally.

263
The regeneration process for rose somatic embryos is affected by many factors. In this 264 process, the formation of embryogenic calli is the key to culturing. This study shows that the 265 leaf can be used to induce embryogenic calli, but the petiole cannot. In addition to the 266 differential expression of genes in different explants, these differences may also be related to

336
The results showed that tilting the culture medium can induce the germination of somatic 337 embryos and increase their overall germination rate. Too much water on the surface of the 338 medium will affect the germination of somatic embryos and the inclined placement of the 339 medium may have caused a certain amount of drought stress, which is conducive to the 340 germination of somatic embryos. The SE2 somatic embryos had the highest germination rate 341 in this study. To enable embryogenic calli to successfully induce somatic embryos and produce 342 more SE2 type somatic embryos, the following factors may need to be considered. The first is

370
The above experiments confirmed that complicated mechanisms exist during the development 371 of somatic embryos, and that many factors can affect them. However, to date, it has not been 372 possible to comprehensively study the internal molecular mechanism and this requires further

555
/ initial value of somatic cells in each group. In the inclined medium, the initial value for SE0 556 was 286, SE1 was 175, SE2 was 67, the secondary embryo was 56 and the polycotyledon 557 was 4. In the medium that was placed flat, the initial value for SE0 was 302, SE1 was 196, SE2 558 was 70, the secondary embryo was 32 and the polycotyledon was 2.

582
Healthy seedlings cultured in SP medium for 16 weeks (E) and rose seedlings transplanted to 583 the greenhouse after rooting (F). Culture conditions: 16 hours for white light/8 hours in 584 darkness at 25 ± 2°C. Scale bar for A-D is 2 mm, and for E, F it is 2 cm. 585 586