Pollen Morphology, Vitality and Storage Capacity of Camellia rosthorniana

Xu Lin^1,2*, Du Kebing³, Chen Fazhi^1,2, Yang Shoukun^1,2, Chen Weidong^1,2

¹Scientific and Research Institute of Forestry and Fruit Sciences of Wuhan City，Wuhan 430075，China
² Landscape Plants Engineering Technology Research Center of Hubei Province，Wuhan 430075，China
³ College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China
* author for correspondence, e-mail: xulin.xulin@yahoo.com.cn

 Introduction

  Fig.1 Flowering plant of C. rosthorniana

The species Camellia rosthorniana, which belongs to Sect. Theopsis of genus Camellia, is an excellent species with fragrant and dense flowers, which could be useful material to cross with other camellias. Therefore, the hybridization of C. Rosthorniana is significant for breeding fragrant cultivars.

We found wild plants of C. rosthorniana at Tianzi Mountain when we surveyed the vegetation of Wuhan City in 1999 (Fig. 1-2). Then we selected a superior individual named ‘Tianshan fen’ from these wild plants and grew it on for ten years. This research focused on the pollen morphology，vitality and storage capacity of C. rosthorniana ‘Tianshan fen’, in order to much understand the pollen background of C. rosthorniana.

Fig. 2  The characteristics of Camellia rosthorniana

A. Canopy B. Flowerclose-up 
C. front of twig D. Back of twig 
E. Upper surface of leaf F. Lower surface of leaf G. flower buds 
H. Roots I. seeds

Materials and methods

Materials

3-year-old strong plants of C. rosthorniana ‘Tianshanfen’ were used as materials, and the experiments performed in Wild Plants Resources nursery of Scientific and Research Institute of Forestry and Fruit Sciences of Wuhan City.

Observations of the pollen morphology under SEM

Flowers were collected in full-bloom stage in mid-March, and the anthers were fixed by 2.5% pentadialdehyde. After dehydration, substitution, CO₂ critical point drying, and coating by ion sputtering equipment, observations were made under JSM-6390/LV scanning electronic microscope, and then photographed.

20 pollens were selected randomly for observations of their shape, polar view, equatorial view, germinal furrow, and exine ornamentation. The length of polar axis, equatorial axis and germinal furrow were measured.

Pollen viability test

Flowers were collected at about 10 a.m. on sunny days when they were just blooming at the full flowering stage. Anthers were collected and placed on white papers for 24 h at room temperature until they ruptured naturally. Pollens were gathered for the test of vitality and storage capacity. 

Determination of pollen germination time

Five kinds of liquid media (Table 1) were dropped on the fluted slide and the fresh pollens were evenly spread on the surface of the medium by using a dissecting needle. Then the slides were placed in culture dishes covered with wet filter paper and cultivated in an incubator for 2, 24 and 48 hours at the temperature  (25 ± 1) ℃ in the dark. Pollen germination rates were observed and calculated to determine the optimal observation time of pollen germination.

Determination of the optimum pollen germination medium

Five kinds of liquid media (Table 1) with the basic ingredients of 200 mg L^-1 MgSO₄ + 100 mg L^-1 KNO₃ + 300 mg L^-1 Ca(NO₃)₂ were designed, supplemented with different concentrations of H₃BO₃ and sucrose, pH 5.5 ~ 6.0. To determine the best pollen germination medium composition for C. rosthorniana, the germination rate under microscope and pollen tube length were measured when cultured for 48 hours under dark conditions.

Every treatment was performed in three replicates while each replication contains five randomly observed horizons when gathering germination rate statistics. The number of pollens in each horizon was more than 50. When the pollen tube length was more than the diameter of pollen, the germination status was recorded. Germination rate (%) = the number of germination pollens in one horizon / total number of pollens in the horizon ×100, taking the average as the pollen germination rate. When measuring the pollen tube length, three horizons were randomly observed, and then taking the average of 20 germination pollens randomly selected in each horizon as pollen tube length.

Determination of pollen storage capacity

Fresh pollens were placed indoors for 24 hours, and then collected in tubes, which were plugged by cotton wool, placed in a desiccator, and then were stored under the condition of 4 ℃. Every 30 days during storage, some pollens were taken out and allowed 5-6 hours to re-absorp water at room temperature for 5-6 hours before measuring the viability of the pollens.

Data analysis

ANOVA analysis and multiple comparisons (Duncan's) were performed with SAS 8.1 statistics packages.

Results and analysis

Observation of pollen morphology

The SEM results showed that the pollen grain of ‘Tianshan fen’ was in the shape of flat spherical, 29.13 um×40.33 um，which greatly differs from the long round shape of other species of Sect. Theopsis (Li et al., 2005). The equatorial view showed broadly oval, and the polar view showed equilateral triangle (Fig. 1 A, B).

The surface of the pollen grain was ornamented with transition from reticulation to foveolation. Short and blunt spikes uniformly grew on coarse and bending murus. The luminas were small, and the size and shape were irregular (Fig. 1 C). The above characteristics were similar to the other six species of Sect. Theopsis, especially to C. euryoides, which differed only in the short and blunt spikes on the surface of pollen grain of ‘Tianshan fen’.

The pollen grain had 3 colporates with smooth colpus membrane, 10 um in length and 50 um in width. The apocolpium area was narrower than other species of Sect. Theopsis. The aperture had a rectangular aperture cap with protein particles, and cap length and width was about 15um and 10um, respectively (Fig.3 B, D). Based on the reports of Li et al. (2005), there was no aperture cap on the other six tested species of Sect. Theopsis, which was very different with ‘Tianshan fen’.

 Fig.3  Pollen morphology of Camellia rosthorniana ‘Tianshan fen’ under SEM
A. Equatorial view; B. Polar view; C. Sculpture; D. Cap of aperture

In vitro culture of pollens

Pollen germination time

With time, the pollen germination rate of all five kinds of media increased (Table 1). Cultured for 2 h, the germination rate on the 1 to 5 media reached to 84.91%, 68.19%, 50.28%, 63.85% and 67.59% of the germination rate after 48 h, respectively, which indicated that a large number of pollen grains had germinated at the beginning of culture. After 24 h, the germination rate on the 1 to 5 media reached to 87.93%, 90.58%, 89.43%, 88.39% and 95.06% of that after 48 h, which indicated that almost all of the pollens had germinated after 24 h culture. Thus, the pollen germination rate after 24 hours in vitro culture was close to the maximum germination rate of 94.28% after 48h culture. Therefore, the pollen germination rate could be recorded after 24 h culture.

Pollen germination medium

After being cultured for 2 h, the pollens germination was slightly restrained by higher concentration of H₃BO_3. But the effect of restraint faded with as time passed. After being cultured for 24 h and 48 h, the germination rate was not significantly different between different concentrations of H₃BO₃ (Table 1). In addition, after being cultured for 48 h, the pollen tube length decreased along with the increasing concentrations of H₃BO₃ which indicated that high concentrations of H₃BO₃ have a certain inhibition effect on elongation of the pollen tube (Table 1). 

Cultured for 2 h, the pollen germination was promoted by higher concentrations of sucrose. The effect was strengthened as time passed by. After 24 h and 48 h, the germination rate was significantly different among various concentrations of sucrose (Table 1). The germination rate was 89.62% and 94.28% in 200 g L^-1 sucrose concentration after cultured for 24 h and 48h, respectively, which was significantly higher than other sucrose concentrations (Table 1). Furthermore, high concentrations of sucrose promoted the elongation of the pollen tube significantly. After 48h, the pollen tube length was with 200 g L^-1 > 150 g L^-1 > 100 g L^-1 (Table 1) , which demonstrated that the higher sucrose concentration (200 g L^-1) was more suitable for pollen in vitro culture of C. rosthorniana ‘Tianshan fen’.

In five kinds of media, the highest pollen germination rate was obtained with the combination of 100 mg L^-1 H₃BO₃ and 200 g L^-1 sucrose, which was up to 94.28%, which was the most suitable medium for pollen germination of C. rosthorniana ‘Tianshan fen’.

Table 1 Pollen germination rate of C. rosthorniana on different concentrations boric acid and sucrose.

Note: Different letters in germination rate and pollen tube length indicate a significant difference（p ＜ 0.05）

Vitality variation of storage pollens

With passage of time, the storage pollen germination rate decreased gradually (Table 2). After storage for 30 d or 60 d at 4 ℃, the pollen germination showed slight decrease, from 94.28% of fresh pollens down to 87.95% and 85.28%, respectively. However, the pollen germination rate decreased greatly and fell to 51.27% when stored for 90 d. Therefore, dried pollens of C. Rosthorniana ‘Tianshan fen’ after 60 d storage under 4 ℃ still had good vitality，which could be used in solving the problem of asynchronous flowering time with C. japonica in cross breeding.

Table 2 Pollen germination rate of C. rosthorniana during storage under 4 ℃

Note: Different letters indicate a significant difference（p＜0.05)

Discussions

As male productive cell carrying genetic information, pollen morphology had higher conservation, and the morphology, including in pollen shape, outline, sculpture, number, location, characteristics of apertura, was an important basis for species identification (Lan and Xu, 1996). Great differences among numerous species of genus Camellia resulted in a great deal of divergences. Therefore, it is reliable to utilize pollen morphology as a method of studying taxonomy of the genus Camellia. At present, study on pollen morphology about C. rosthorniana had not been reported. In the present research, results showed that pollen shape of C. rosthorniana ‘Tianshan fen’ was in accordance with reported general characters of genus Camellia (Wei et al., 1992; Wang et al., 1995; Wang et al., 1997). However, there were great differences between C. rosthorniana with the other reported six species of Sect. Theopsis (Li et al., 2005) in pollen shape, polor view, colporates, colpus membrane, apocolpium area, aperture cap and sculpture. These differences showed that C. rosthorniana might have special taxonomy status in genus Camellia and further research was needed.

Pollens viability measurement has many methods, the in-vitro germination method was used widely for its accuracy and convenience. A large number of studies showed that optimum concentrations of H₃BO₃ and sucrose was beneficial for pollen germination and tube elongation (Guo et al., 2002; Wang and Zhang, 2004；Kakegawa et al., 2005；Nian et al., 2005；Du et al., 2007). In this study, in in-vitro pollengermination of C. rosthorniana ‘Tianshan fen’, the optimum concentrations of H₃BO₃ and sucrose were 100 mg L^-1 and 200 g L^-1, respectively. This result exhibited some differences to C. fraterna in germination time and concentrations of H₃BO₃ and sucrose (Tang and Chen, 2004), which resulted from of interspecific differences and in-vitro culture surroundings.

Suitable storage of pollens could ensure obtaining a greater quantity of progenies during hybrid breeding (Parton et al., 2002; Wang et al., 2009). In this study, storage for 60 d at 4℃, the pollen germination rate of C. rosthorniana ‘Tianshan fen’ was still more than 80%, which was similar to some species (Wang et al., 2009). However, in the next 30 days of storage, the pollen germination rate rapidly decreased to about 50%, which would reduce the population quantity progenies. In general, the flowering stage of C japonica was from February to April, and C. rosthorniana ‘Tianshan fen’ from February to March. Therefore, the pollens of ‘Tianshan fen’ storage for 30-60 d could solve the problem of asynchronous flowering time.

References

Du Kebing,Shen Baoxian,Xu Lin,Tu Bingkun,Cai Chongfa,Dai Miaohong. 2007. Changes of viability of stored poplar pollens and its feasibility for cross breeding. Journal of Huazhong Agricultural University26 (3)385–389.
Guo Guangming，Zhang Fusuo，Shang Zhonglin，Zhang Ximei. 2002. Effects of boron on cytosolic Ca²⁺ in germinating pollen grain cells of Lilium davidii Dutchartre. Journal of China Agricultural University，7 (5)：32–37.
Kakegawa K，Ishii T，Matsunaga T. 2005. Effects of boron deficiency in cell suspension cultures of Populus alba L. Plant Cell Rep，23：573–578.
Lan Shengyin，Xu Zhenxiu. 1996. Stripping observation and illustration of plants pollen under scanning electronic microscope. Beijing：Science Press.
Li Guangqing，Sun Li，Liu Yan. 2005. Pollen features of 6 species in Sect. Theopsis of genus Camellia. Journal of Tropical and Subtropical Botany，13 (1)：40–44.
Nian Yuxin，Luo Fengxia，Zhang Ying，Sun Xiaomei，Zhang Li. 2005. Studies on the culture solution for lily pollen vitality test. Acta Horticulturae Sinica，32 (5)：922–925.
Parton E，Vervaeke I，Delen R，Vandenbussche B，Deroose R，Proft M D. 2002. Viability and storage of bromeliad pollen. Euphytica，125：155–161.
Tang Qianrui，Chen Youyun. 2004. Study on the optimal medium in the pollen germination of Camellia fraternal. Journal of Hunan Forestry Science and Technology，31 (1)：30–32.
Wang Chao，Zhang Guiling. 2004.The preliminary research on cabbage pollens sprouting in vitro. Acta Horticulturae Sinica，31 (2)：209.
Wang Fuxiong. 1995. The pollen morphology of plants in China. Beijing：Science Press.
Wang Kaifa，Wang Xian-zeng. 1983. An introduction to palynology. Beijing：Peking University Press.
Wang Renxiang，Hu Changhua，Liang Qianhua，Zhao Ruifeng，Liang Shengye. 1997. The pollen of Sect. Chrysantha plants studied by scanning electron microscope (Ⅰ). Journal of Guangxi Plants，17 (3)：242–245.
Wang Wenli，Zhao Lanyong，Zhu Cuiying，Zheng Chengshu，Zhang Youpeng，Geng Cuifang. 2009. Studies on pollen viability and its storage conditions of Pingyin rose. Acta Horticulturae Sinica，36 (4)：593–598.
Wei Zhongxin，Zavada M S，Min Tianlu. 1992. Pollen morphology of Camellia（Thuaceae）and its taxonomic significance. Acta Botanica Yunnanica，14 (3)：275–282.