Yuping Ll，F(xiàn)engxia LUO，Yunjie SHENGJinling Institute of Technology College of Horticulture，Nanjing 210038，China

Characteristics of Pollen Germination and Storage of Zygocactus truncates

Yuping Ll，F(xiàn)engxia LUO*，Yunjie SHENG
Jinling Institute of Technology College of Horticulture，Nanjing 210038，China

The research，based on pollens of Zygocactus truncates during full-bloom period，selected suitable culture medium and temperature for pollen germination and explored the effects of storage temperatures and pollen collection methods on pollen vitality of Zygocactus truncates and the results showed that the culture medium containing 200 g/L saccharose，20 mg/L boric acid，and 20 mg/L calcium chloride is suitable for pollen germination of Zygocactus truncates；when the temperature kept below 20℃，pollen germination period last short，and germination rate kept higher；pollen germination performed better with filaments，and pollen tube grew well.

Zygocactus truncates；Pollen germination；Fluid medium；Storability

Z ygocactus truncates，endemic to Brazil，has leafless green stems and the stems are composed of strongly flattened segments，having“teeth”of varying shapes along their edges and at the ends. The flowers are held at a constant angle above the horizontal with the higher side different from the lower side and of various colors，including red，orange，pink and white［1］.In Germany and America，Zygocactus truncates is cultivated and produced in a large scale，and it is one of major potted flowers in winter.In the 1980s，Zygocactus truncates was introduced to China and is produced in a small scale in Tianjin，Dalian，Qingdao，Shanghai，Guangzhou，and Xiamen.Pollen is part of the male gametophyte of higher plants，delivering male genetic materials at sexual propagation and constituting important materials of sporopollen analyzing，swarm cultivation and drug manufacturing［2-3］.The research，based on pollens of Zygocactus truncates during full-bloom period，selected suitable culture medium and temperature for pollen germination and explored the effects of storage temperatures and pollen collection methods on pollen vitality of Zygocactus truncates in order to provide references for hybridization of Zygocactus truncates.

Materials and Methods

Materials

The pollens of Zygocactus truncates during full-bloom period were taken as test materials.

Methods

Selection of liquid medium for in vitro pollen germination

The test designed 8 saccharose concentrations，involving 0，50，100，150，200，250，300 and 350 g/L in order to select a concentration suitable for pollen germination.On basis of simple factor design of experiment，boric acid and calcium chloride were added as per orthogonal experimental design （Table 1）to discuss liquid medium for pollen germination of Zygocactus truncates.Specifically，fresh pollen was collected during 08:00-09:00，added into a glass tube，and well oscillated.Then，pollen was scattered on nutrient solutions and well mixed with the solution.It is notable that every nutrient solution was repeat-ed for four times.Subsequently，a glass slide was placed in a petri dish，which was placed in a room at 25℃and observed once every 2 h.The final measurement time started when twice measurements of germination rates kept basically the same.For every repetition，100 pollens would be observed and the number of germinated pollen was computed as per the standard that the length of pollen tube exceeded diameter of pollen grain，as follows:

Germination rate=The number of germinated pollen grain/The number of pollen×100%.

Effects of cultivation temperature on pollen germination time After determination of the suitable culture medium of in vitro germination of pollen，pollens were then placed in an incubator at 15 and 20℃.The test was observed once every 2 h and changed to once every one hour after pollen germination，until the observed data changed insignificantly.Still，the measurements of pollen germination rate were the same as the equation above.

Effects of storage temperature on pollen germination time At the beginning，loose pollens were collected and dried in the shade.Then，pollens were put into a glass tube which was labeled and placed in the shade and then a refrigerator at 4 and-20℃，respectively.It is notable that pollen germination rate was observed once every two days until pollen germination activity was below 5%.The nutrient solution contained 200 g/L saccharose，20 mg/L boric acid，and 20 mg/L calcium chloride，and the culture temperature was 20℃.In addition，the measurements of pollen germination rate were the same as the equation above.

Effects of pollen collection methods on pollen germination activity after storage The dehiscent anthers and anthers with 1 cm filaments were put into a glass tube and stored at 4℃.Pollen germination rate was measured once every 4 days.The anther culture solution contained 200g/L saccharose，20 mg/L boric acid，and 20 mg/L calcium chloride，and the culture temperature was 20℃.In addition，the measurements of pollen germination rate were the same as the equation above.

Results and Analysis

Effects of fluid medium on pollen germination

Effects of saccharose concentration on pollen germination When pollens were cultured with saccharose at different concentrations，pollen would swell.After 3 h，pollen tubes germinated and grew from germinal furrows.After 25 h，the germination process ended and pollen germination kept almost the same after 29 h.As shown in Table 2，when saccharose concentration was 250 g/L，pollen germination rate reached the peak at 8.5%，and the rates of rest treatments generally maintained lower.

The orthogonal test of culture medium of in vitro germination of pollen As shown in Table 3，pollen germination rate achieved the peak at 35.9%in the culture medium containing 200 g/L saccharose，20 mg/L boric acid，and 20 mg/L calcium chloride and the effects from high to low were boric acid＞saccharose＞calcium chloride.It is notable that highly-concentrated saccharose would prevent pollen germination，but suitable boric acid and calcium chloride would promote pollen germination.The optimal culture solution can be concluded containing 200 g/L saccharose，20 mg/L boric acid，and 20 mg/L calcium chloride.

Effects of culture temperature on germination time

As shown in Table 4，pollens germinated earlier in an incubator at 20℃，with a higher germination rate. Specifically，pollens germinated 2 h after cultured in an incubator，which was 1 h earlier compared with the situation at 15℃.After 29 h，pollen germination ended，which was 3 h fewer compared with that at 15℃.

Effects of storage temperature on pollen germination rate

It can be concluded from Fig.1 that pollen germination rate dropped fast at room temperature and the rates were 30%，23%，and 18%after 1，2 and 3 d storages.At 4℃，the germination rate was 14%5 d after storage，8%9 d after storage and below 5%12 d after germination.It can be concluded that pollen of Zygocactus truncates can be stored for 12 d at 4℃.After 3 d storage at-20℃，germination rate of pollen was 18%，13%after 5 d storage and 10%after 10 d storage.

Table 1 The orthogonal experiment of pollen germination of Zygocactus truncates with liquid medium

Table 2 Effects of saccharose concentration on pollen germination rate

Effects of pollen collection methods on germination rate

As shown in Fig.2，to collect pollen with filament extended storage periods of pollen and the germination rate still kept over 5%after 15 d storage.What’s more，with pollen stored for 9 d with filament，the number of germinated pollen would be much more and pollen tubes grew well.Incontrast，the number of germinated pollen was small at collecting pollen without filaments.

Table 3 The orthogonal test results and range analysis of fluid medium for pollen in vitro germination

Table 4 Effects of culture temperatures on pollen germination time

Conclusions and Discussions

The research indicated that the culture medium containing 200 g/L saccharose，20 mg/L boric acid，and 20 mg/L calcium chloride is suitable for pollen germination of Zygocactus truncates. Specifically， saccharose plays the role of supplying energy and adjusting osmotic pressure during pollen germination［4］.Highly-concentrated saccharose would prevent pollen germination，which coincided with the conclusion of Zhang et al.and He et al.［5-6］.Furthermore，boric acid at a suitable concentration would promote pollen germination and pollen tube growth［7-10］.The research suggested that pollen germination rate reached the highest with boric acid concentration at 20 mg/L，possibly caused by the complex formed by boric acid and saccharose，advancing sugar absorption and metabolism［11-12］. On the other hand，temperature also has effects on pollen germination. Jiang et al.believed that the temperature suitable for pollen germination and growth is in the range of 25-30℃，and the temperature of 30℃ is optimal，when pollen germination rate and pollen tube length both reached the highest.In contrast，with temperature below 20℃ or higher than 35℃，pollen germination rate was lower and pollen tube grew slowly［13］.When the temperature kept below 20℃，pollen germination period last short，and germination rate kept higher.Therefore，the temperature at 20℃is suitable for Zygocactus truncates growth.It is clear that low temperature would reduce pollen respiration and other physiological functions，which is conductive to long-term maintenance of pollen vitality，because low temperature reduces respiration strength and enzymatic activity［14］.Sun et al.believed that frozen storage of lily pollen performed the best at 4℃［15］.The research indicated that germination rate of pollen of Zygocactus truncates was decreasing at room temperature and can be stored as many as 5 d，10 d at 4℃，and 8 d at-20℃.It is notable that pollen with filaments germinated well and pollen tube grew well，possibly caused by nutrients supplied by filaments for pollen，extending pollen death.

References

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蟹爪蘭花粉萌發(fā)及貯藏特性

李玉萍，羅鳳霞*，盛蕓潔 （金陵科技學(xué)院園藝學(xué)院，江蘇南京 210038）

以蟹爪蘭花粉為材料，采用液體培養(yǎng)法研究培養(yǎng)基組成、培養(yǎng)溫度、花粉采集方式、貯藏溫度等對(duì)花粉萌發(fā)特性的影響。結(jié)果表明：適宜蟹爪蘭花粉萌發(fā)的液體培養(yǎng)基為：200 g/L蔗糖+20 mg/L硼酸+20 mg/L二氯化鈣；20℃下蟹爪蘭花粉萌發(fā)時(shí)間比較短，且萌發(fā)率較高；有花絲的花粉萌發(fā)較好，花粉管生長(zhǎng)良好。

蟹爪蘭；花粉萌發(fā)；液體培養(yǎng)基；貯藏性

江蘇省高校自然科學(xué)研究項(xiàng)目（編號(hào)：10kjd210005）。

李玉萍（1976-），女，甘肅酒泉人，博士，副教授，主要從事園林植物遺傳育種和應(yīng)用研究，E-mail：lyp@jit.edu.cn。*通訊作者，教授，E-mail：luofx@jit.edu.cn。

2015-01-01

2015-02-13

Supported by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China（10kjd210005）.

.E-mail:luofx@jit.edu.cn

January 1，2015Accepted:February 13，2015