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A novel in vitro germination method revealed the influence of environmental variance on the pecan pollen viability
Scientia Horticulturae 181 (2015) 43–51
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A novel in vitro germination method revealed the influence of environmental variance on the pecan pollen viability Hua-Zheng Peng 1 , Qun-Ying Jin 1 , Hua-Lin Ye, Tang-Jun Zhu ∗ Zhejiang Provincial Key Lab of Bamboo Research, Zhejiang Forestry Academy, Hangzhou 310023, China
a r t i c l e
i n f o
Article history: Received 25 August 2014 Received in revised form 10 October 2014 Accepted 30 October 2014 Keywords: Pecan pollen Pollen germination Pollen rehydration Pollen tube growth Pollen viability
a b s t r a c t Pecan is one of the most important horticultural nut crops in the world. Germination tests are vital to the safe application of artificial pollination and cross breeding of pecan. Although many in vitro germination methods have been proposed, the realization of robust and reproducible methods to induce morphologically normal growth pollen tubes in vitro for detailed dynamic observation has often proved challenging in pecan. We employed a mixing and spreading method to realize the uniform distribution and germination on agarose medium, which resulted in the unprecedented level of germinational repeatability and observability. With this method, we investigated the influence of environmental variance on the in vitro germination of pecan pollen. The data from germination rate, burst rate, pollen tube length and germination speed indicated the pecan pollen had an optimal germination temperature at 25 ◦ C but optimal rehydration temperature at 16 ◦ C which was lower than minimum germination temperature. And this optimal rehydration temperature was harmful to pollen germination if pollen was ready to germinate after rehydration. It helped researchers understand the mechanism of probable promotion of high day/low night temperatures to pollen germination in nature. Results further revealed that pecan pollen didn’t lose their viability rapidly in first several days at ambient conditions but changed to a different rehydration and germination condition. In addition, we proposed here a new germination speed parameter GT50 to indicate the early change of pollen viability. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Pecan (Carya illinoinensis), belonging to the Juglandaceae family, originally grows in south-central North America and is one of the most important horticultural nut crops in the world (Wetzstein et al., 1996). It is a wind-pollinated monoecious plant with staminate flowers organized into an ament or catkin and female flowers borne on a spike (Wetzstein and Sparks, 1986). Because of the heterodichogamy and short flowering period, breeding programs often have to use stored pollen to perform particular pecan crossing. Efficient and reproducible viability testing before crossing is essential to avoid the use of nonviable stored pollen, or it will result in the loss of the cross for the year (Sparks and Madden, 1985). Germination tests have generally been considered to be the best in vitro indicator of pollen usefulness, which assess the viability of pollen samples by germinating a sample of pollen grains in an artificial media (Galleta, 1983). Previous studies have proved that the in vitro
∗ Corresponding author. Tel.: +86 571 87798231; fax: +86 571 87798113. E-mail address: [email protected] (T.-J. Zhu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.scienta.2014.10.057 0304-4238/© 2014 Elsevier B.V. All rights reserved.
germination of most pollens are mainly affected by the osmotic potential regulator (such as sucrose), concentration of borate and so on in medium although the effects of these ingredients in medium are not completely understood (Holdaway-Clarke et al., 2003). In pecan, trials have proved that fresh pollen can germinate in an artificial medium (Wetzstein and Sparks, 1985; Yates et al., 1986) and controlled rehydration of dry pollen before placing in the germination media is vital to determining accurate germination rate (Yates and Sparks, 1989). The rehydration experiment of dry pecan pollen helps prove that pollen stored for nearly 2 years at −80 ◦ C or −196 ◦ C, but not −10 ◦ C, can retain germination capacity equal to freshly collected pollen by both in vitro germination test and fruit set analysis (Yates and Sparks, 1989). Further studies reveal that pecan pollen maintained at −12 ◦ C for 2 years is as viable as freshly collected pollen if it is oven dried at 35 ◦ C to a constant weight and stored in moisture-proof bags (Yates et al., 1991). Some experiments even reveal that the viability of pecan pollen does not decline in frozen storage up to 13 years (Sparks and Yates, 2002). On the other hand, researchers also find that pecan pollen loses their viability rapidly in first several days although it can retained the low germinational capacity for 59 days at ambient laboratory conditions (Yates and Sparks, 1989). It is surprising that pecan pollen will
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lose its viability rapidly in short time since this type of desiccationtolerant pollen can survive for a long period of time after anthesis. In addition, when it is introduced from its original region into other climate zone, pecan may encounter some problems in pollination and germination although the seedlings still grow well (Peng et al., 2012). To deeply explore the detailed influence of environmental variance on pollen viability, we need a high robust and reproductive method to induce in vitro pollen germination and morphologically normal growth of pollen tube. But the designing of such method in pecan is always a challenging. In recent years, a new cellophane based method is proposed to improve the germination rate and repeatability of pecan pollen, which has considerable utility compared to the traditional drop, tube, multi-well liquid and multi-well agar methods (Conner, 2011). However, there still exist some obvious defects in current method. First, non-replicable results still exist due to the use of ‘dusting’ method to spread pollen on medium surface. Pollen density can influence the germination of plant pollen no exception of pecan pollen whose germination proves decreasing below or above the optimal concentration (Yates et al., 1986). Second, the current method can’t be used to observe the germination process directly and dynamically. The advancing of novel method in model plant Arabidopsis thaliana using agarose and cellophane as support greatly facilitates efficient, reproducible studies of the post-hydration development of pollen from A. thaliana and its relatives (Rodriguez-Enriquez et al., 2013). But, for in vitro germination of pollen from pecan and its relatives, a robust and reliable germination method, through which germination process of differently treated pollen can be compared in detail, has remained elusive. In this paper, we proposed a novel method for efficient in vitro germination with very high repeatability which can not only promote germination and tube growth, but also realize dynamic observation of germination process. We used this system to evaluate the influence of environmental variance on in vitro germination of pecan pollen. 2. Materials and methods 2.1. Collection and use of pollen Studies on the pollen germination were conducted using ‘Pawnee’ pollen collected in early May of 2013. Pollens were collected, dried and stored according to the reference (Conner, 2011). They were aliquoted into 50-ml polypropylene centrifuge tubes and frozen at −80 ◦ C until use. Germination tests were conducted using newly thawed and RT-stored pollen samples. For a newly thawed pollen sample, a 50-ml sample tube was taken out of the freezer and immersed in ice to thaw for 2 h and then transferred to dessicator at room temperature for 2 h. For RT-stored pollen sample, the above-mentioned sample tube was finally stored at dessicator at room temperature for 3d or 7d before germination tests. 2.2. Pollen culture medium and apparatus Germination medium was mainly made on the base of previous studies with some necessary optimization for the method in this paper (Conner, 2011). It consisted of 20% sucrose, 4 mM Boric acid, 3 mM CaCl2 , 0.67 mM KCl, 10 mM MES-Tris (pH5.8) and 1% agarose (Biowest, Spain). The liquid part of the germination medium was made by dissolving sucrose first and then adding the other materials except agarose. The liquid part was used here to mix rehydrated pollen for spreading. For the final germination medium, the appropriate agarose was added to the above solution and briefly heated in a microwave. Each 5 ml medium was then poured into 5 cm petri dish before its solidification and blown openly in clean bench for
10 min. The plate can be stored at 4 ◦ C temporarily for a couple of days before use. 2.3. Pollen germination method Germination tests here included several necessary steps (Fig. 1A). First, the thawed pollen was rehydrated for hours before transferring to germination medium. In this step, pollen samples were dusted into petri dishes and suspended above water in a sealed container at required rehydration temperature. Second, the rehydrated pollen was weighed into 1.5 ml centrifuge tube and then fully mixed with liquid germination medium in proportion by tip. Here 10 l suspension was dropped into the germination medium plate for a single test right after the mixing. Third, the pollen suspension was spread evenly on plate using cell spreader (1 cm side length) by a unidirectional spreading. Each germination plate was immediately covered after spreading to prevent drying. Finally, the plate, suspended above water in a sealed box, was incubated in the dark at certain temperature to germinate for at least 24 h. When taken for examination every 3 h, the plate was covered and observed directly by inverted microscope. Each treatment of every sample was repeated three times. 2.4. Microscopy and data analysis During the germinational process, the germinational phenomena in all tests were observed and recorded by Olympus inverted fluorescence IX71 microscope with DP71 camera at 100× magnification. A pollen grain was considered to have germinated if the pollen tube was equal to or greater in length than the width of the pollen grain. And a pollen grain was considered to be burst if the inclusion of pollen diffuse. The grain both have germination and burst (usually burst from the pollen tube) will be considered as germination instead of burst. Germination images were further measured including count and pollen tube measurement by Image Pro Plus 6.0. In each replication, three vision fields were examined with each field containing 200 or so pollen grains. The data were exported to Excel software for further analysis including arithmetic mean and error analysis. The arithmetic mean of data from three vision fields was taken as one experimental repeat. For length measurement of pollen tube, 50 pollen tube or all (if the quantity was not enough) was measured from each of three vision fields and their arithmetic mean was taken as one experimental repeat. Experimental errors were estimated through data from three repetition. Paired t-test was performed in Excel for group data comparison and all indicated P values were two tailed. 3. Results 3.1. The selection strategy of rehydration and germination condition We used ultra cold stored pollen samples instead of freshly collected pollen samples in our experiments, which is on one hand because of better repeatability and convenience, on the other hand because of equal germination capacity to freshly collected pollen by both in vitro germination test and fruit set analysis (Sparks and Yates, 2002; Yates et al., 1991; Yates and Sparks, 1989). In our preliminary experiments, we used the mixing and spreading method to make sure the optimal pollen concentration for high germination rate, which was about 0.02 g rehydrated pollen per 100 l liquid germination medium for the sample we used. Our studies also indicated that the pollen could hardly germinate below 22 ◦ C which was then set as the temperature boundary between rehydration and germination. We also noticed from the climate record in pecan’s naive region that the day mean minimum temperature
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Fig. 1. Flow chart and corresponding effect of the novel germination method of pecan pollen in vitro. (A) The flow chart of germination method. The solid line stands for the real operation in this paper while the dotted line indicates the future use. (B) Pecan pollens spread in 5 cm Petri dish. (C) Germination observation of pecan pollen in the abovementioned B under inverted microscope. The white arrows indicate burst. (D) Germination result of pecan pollen using cellophane-agarose method (Rodriguez-Enriquez et al., 2013). Bar = 100 m.
was 10 ◦ C or so and the day mean maximum temperature was about 31 ◦ C during the flowering season. Then the scope of rehydration temperature was set from 4 ◦ C to 22 ◦ C while the scope of germination temperature was set from 22 ◦ C to 31 ◦ C. The 4 ◦ C was chosen for the reason that this temperature is widely used in the cold treatment and temporary storage of biology sample. For rehydration time, 4 h was selected as standard while the double time (8 h) was chosen as prolonged time according to the references and our preliminary experiments (Conner, 2011). The effect of cellophane method which has been successfully used in A. thanliana (Rodriguez-Enriquez et al., 2013) was compared to the present method, indicating the obviously better uniformity and visuality of method proposed here (Fig. 1).
3.2. The proper low rehydration temperature promoted pollen germination The newly thawed pecan pollen was used to estimate the influence of rehydration temperature and germination temperature on germination. The data indicated that germination rate rose with the increase of rehydration temperature only when the rehydration temperature was below 16 ◦ C no matter how long the rehydration time was (Fig. 2A and C). The comparison of
germination results between 16 ◦ C and 22 ◦ C rehydration temperature could be further analyzed by statistical method (Table 1), indicating the significant improvement at 16 ◦ C rehydration condition for no RT-stored pollen samples. And the germination rate rose with the increase of germination temperature only when the germination temperature was below 25 ◦ C. While the pollen burst rate mainly rose with the increase of germination temperature from 22 ◦ C to 31 ◦ C and less affected by the rehydration temperature (Fig. 2B and D). Thus the best germination result with the highest germination rate and comparatively low burst rate occurred at a combination of 16 ◦ C rehydration temperature and 25 ◦ C germination temperature under both standard and prolonged rehydration time. The results definitely showed that the rehydration temperature was comparably important to pecan pollen germination with germination temperature. And the rehydration temperature lower than minimum germinational temperature was beneficial to pecan pollen germination.
3.3. RT storage first changed pollens’ optimal rehydration condition We then investigated the germination results of RT-stored pecan pollens. After being stored at RT for 3d and 7d, the pollen
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Fig. 2. The germinational result of newly thawed (no RT-stored) pecan pollen under different rehydration and germination conditions. (A) The germination rate after 4 h rehydration. (B) The burst rate after 4 h rehydration. (C) The germination rate after 8 h rehydration. (D) The burst rate after 8 h rehydration. All pictures share same data tabs indicated in B.
Table 1 Comparison of germination rates between rehydration temperature 16 ◦ C and 22 ◦ C by statistical test. Pollen sample no RT-stored pollen
Rehydration Temp ( C)
Temp ( C)
Rate (%)
16
22 25 28 31 22 25 28 31 22 25 28 31 22 25 28 31
36.0 50.6 40.1 36.2 30.9 48.6 38.7 33.9 23.3 41.8 35.3 33.3 21.8 37.4 34.1 29.9
0.047*
22 25 28 31 22 25 28 31
17.6 31.3 27.7 25.4 11.2 19.8 25.0 22.3
0.062
16
22
4
16
22
P < 0.05.
P value
4
8
*
◦
Time (h)
22
3d RT-stored sample
Germination ◦
0.043*
samples under standard rehydration time showed similar germination pattern including the appearance of optimal rehydration temperature (P = 0.062, Table 1.) except for the lower germination rates (Figs. 3A and 4A). But it was surprising that under the prolonged rehydration time the germination rate of pollens increased rapidly at rehydration temperature 22 ◦ C as if the ‘sleeping’ viability had been reactivated (Figs. 3C and 4C). And the germinate rate of RT-stored sample was so high that it could reach more than 85% of the highest level of no RT-stored samples when the RTstored sample was reactivated by higher rehydration temperature and prolonged rehydration time (Fig. 3C). On the other side, it was predictable that the burst rate increased with the increase of germination temperature and RT-stored time, especially germination at higher temperature such as 28 ◦ C and 31 ◦ C after longer RT-storage (Figs. 3B and D and 4B and D). Thus we deduced that the pecan pollens mainly changed their rehydration condition instead of losing their viability rapidly during the early days. 3.4. RT storage also changed pollen’s optimal germination condition Germinational data indicated that the optimal germination temperature was 25 ◦ C in no RT-stored and 3d RT-stored pollen samples (Figs. 2A and C and 3A and C), but it slowly changed to 28 ◦ C in 7d RT-stored sample (Fig. 4A and C). And it should be noticed that this optimal germination condition which led to the highest germination rate also led to the higher burst rate (Fig. 4B and D).
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Fig. 3. The germinational result of RT-stored (3d) pecan pollen under different rehydration and germination conditions. (A) The germination rate after 4 h rehydration. (B) The burst rate after 4 h rehydration. (C) The germination rate after 8 h rehydration. (D) The burst rate after 8 h rehydration. All pictures share same data tabs indicated in B.
This situation was further supported by the evidence from pollen tube length which is one of important indicators of pollen viability (Fig. 5). For no RT-stored pollen, the pollen tube lengths were close under almost all rehydration and temperature condition except 8 h rehydration at 22 ◦ C (Fig. 5A and D). For 3d RT-stored group, the difference of pollen tube lengths from different samples became large (Fig. 5B and E). And for 7d RT-stored group, 28 ◦ C had obviously become the optimal germination temperature based on average pollen tube length (Fig. 5C and F). In addition, it was found that too low germination temperature like 22 ◦ C after prolonged rehydration time was harmful to pollen tube growth (Fig. 5D and E).
25 ◦ C but not obviously at high temperature like 28 ◦ C and 31 ◦ C (Fig. 6A–C). The situation was quite different to prolonged rehydration time under which RT-storage can’t obviously increase GT50 of pollen sample (Fig. 6A–C). In addition, it was surprising that RT storage could sharply increase the GT50 of pollen samples rehydrated at 4 ◦ C although it usually less affected the GT50 of pollen samples germinated at high temperature (Fig. 6C). Thus, the evidence from germination time further indicated that the optimal rehydration and germination temperature tended to be higher temperature since the germination ability of pollen samples at lower temperature was more likely to be inhibited during RT storage.
3.5. Germination time also suggested the change of optimal germination temperature in RT-stored pollen
3.6. Cold treatment after rehydration impairs the pollen germination
Besides germination rate and pollen tube length, germination speed is another important parameter to evaluate the pollen germination ability. We here used the median germination time (GT50) as a substitute of germination speed which was defined as the time to reach half of the highest germination rate. The results indicated that pollen GT50 was mainly affected by rehydration time and germination temperature instead of rehydration temperature (Fig. 6). The data showed that higher germination temperature usually led to lower germination time, that was faster germination speed. Under standard rehydration time, RT storage would increase GT50 and the incremental degree depended on germination temperature, that was to say, RT storage can greatly increase GT50 of pollen sample germinated at low temperature like 22 ◦ C and
We have proved proper low rehydration temperature can promote the germination of pecan pollen in vitro (Fig. 2). But if the cold treatment occurs after standard rehydration, what the influence will be? Our data indicated that cold treatment was harmful to germination once the pollen had fulfilled its rehydration except 4 ◦ C treatment (Fig. 7A and D). And the optimal rehydration temperature above-mentioned, that was 16 ◦ C, on the contrary inhibited the pollen germination most. Besides, the low germination rates from cold treatments were accompanied by higher pollen burst rate compared to the germination without cold treatment (Fig. 7B and E). Only the pollen tube length seemed not to be suppressed by cold treatment (Fig. 7C and F). It is understandable that 4 ◦ C treatment didn’t restrict the pollen germination since this temperature
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Fig. 4. The germinational result of RT-stored (7d) pecan pollen under different rehydration and germination conditions. (A) The germination rate after 4 h rehydration. (B) The burst rate after 4 h rehydration. (C) The germination rate after 8 h rehydration. (D) The burst rate after 8 h rehydration. All pictures share same data tabs indicated in B.
can keep the pollen viability temporarily. But the influence of other cold treatments on rehydrated pollen was indeed surprising and it suggested that the low temperature exerts its promotional function only at early stage of rehydration.
4. Discussion 4.1. Advantages of this germination method of pollen in vitro The uniqueness, simplicity and independence of pollen germination as well as its importance to scientific and applied studies of plant reproduction, have created a strong demand for in vitro systems by which pollen germination and tube growth can be induced. Although numerous germination methods have been proposed before, a novel method mainly using cellulose membrane and spermidine is especially attractive, which is applied successfully in the germination and tube growth of A. thaliana pollen in vitro (Rodriguez-Enriquez et al., 2013). But when the aforementioned method in A. thaliana was used in the germination of pecan-like pollens, the observational effect was unacceptable mainly due to the serious uniformity problem and density-dependent pollen germination. In pecan, our own trials found that single liquid method also failed to exhibit the ideal germinational effect although it can distribute pollen grains uniformly in initial mixing step. This is obviously aggravated after pollen tube growth due to the twine of pollen tube, leading to the poor observability (Zhang et al., 2013, 2014).
In this paper, the liquid germination medium was used to thoroughly mix the pollen grains and cell spreader was used to disperse the pollen evenly on the medium surface, which are the two key elements of our method. Although it was very simple, it performed effectively and fully made use of the superiority of pollen system (a well-distributed cell suspension). Compared to the brushing or dusting methods used before, the mixing-spreading method led to much higher uniformity and greatly improved the repetitiveness and reliability of pollen experiments, which proved helpful in elucidating the influence of environmental factors on pollen germination in this paper. The mixing-spreading method also has good compatibility with previous methods, that is to say, if it’s needed, the pollen can also be spread on the cellophane surface or slide surface. And the successive cell biology studies such as dynamic observation and the location of sperm cells can also be performed in the method. It excludes an unnecessary worry that immersing pollen in liquid culture will inhibit its germination ability since this method only involves a transient mixture of liquid medium unlike the traditional liquid germination method. Furthermore, the small amount of liquid spreading with pollen will soon be absorbed by agarose medium since the solid medium has been blown to relatively dry state. In addition, the pollen will not sink into the medium and pollen tube almost can’t grow into the medium since the agarose concentration is relatively higher (1% agarose) instead of 0.5% agarose in A. thaliana method. In other words, these improvements not only can’t restrict the ventilation of germinational environment, but also can lead to adequate touch and even dispersing of pollen on medium, which finally realize the
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Fig. 5. The pollen tube length of differently stored pecan pollens under different rehydration and germination conditions. (A)–(C) The germinational result of pecan pollens after 4 h rehydration. (D)–(F) The germinational result of pecan pollens after 8 h rehydration. (A) and (D) no RT stored pollens. (B) and (E) RT-stored pollens for 3d. (C) and (F) RT-stored pollens for 7d. All pictures share same data tabs indicated in C.
excellent uniformity and direct observation of pollen germination. Finally, as far as germination rate produced by different germination methods is concerned, we think that they can’t be strictly compared by the results from different papers since the pollen samples may come from different cultivars and have other different environmental conditions not mentioned by the authors. But we know that the liquid method has ever been compared with cellophane method, indicating the relatively low germination rate of liquid method (Conner, 2011), which was also supported by our trials with same pecan cultivar. In this paper, the maximum germination rate brought by our method were equivalent to the result from cellophane method (Fig. 1) and the result from previous report (Conner, 2011). Thus, we think that this new method will greatly mine the potential of pollen system in studies of plant reproductive mechanism and in setup of plant cell model. 4.2. The evaluation of the viability of desiccation-tolerant pollen Presently there exist several methods to evaluate the pollen viability including viability stain and in vitro germination, among which the in vitro germination method is thought to be more persuasive (Galleta, 1983). A fixed so-called “optimal” condition is usually used in the determination and comparison of pollen viability. Through our experiments, we found in pecan the rehydration condition was one of the keys to understand the durable ability of pollen germination. That is to say, pecan pollen actually doesn’t lose their viability rapidly but move to a different state in first several days. If we continue using the initial experimental condition
including the rehydration condition to test pollen viability, the germination result is much worse than the real value. And this also reminds us of cautious using of in vitro germination method to compare pollen viability. In plants, it has proved that controlled rehydration allows the phospholipids in the plasma membrane bilayer to go through phase transition before being exposed to bulk water (Crowe et al., 1989). Thus the change of rehydration condition probably suggest the transition of physiological state of plasma membrane which is the mechanism of early change of pollen viability. There are also some other important parameters such as pollen tube length and germination speed to evaluate the pollen viability besides the germination rate during in vitro germination test. Pollen tube length is seldom mentioned in previous studies of pecan pollen probably due to the use of unstable method although it is widely used in many other species as one of important indicators to determine pollen germination. Based on this new germination method, we found pecan pollen tube length was quite different from the germination rate on the sensitivity to temperature. While the germination rate is obviously influenced by both rehydration and germination temperatures, the pollen tube length is mainly influenced by germination temperature which was obvious for RT-storage samples (Fig. 5B, C, E and F). And this effect of germination temperature on pollen tube length was not suppressed by burst since high germination rate at high temperature sometimes was accompanied by high burst (Figs. 4B and D and 5C and F). But there are also some deficiency in the current germination process when taking the pollen tube length as an indicator of pollen viability since the growth of pollen tube seemed more
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Fig. 6. The median germination time of differently stored pecan pollens under different rehydration and germination conditions. (A)–(C) The germinational result of pecan pollens after 4 h rehydration. (D)–(F) The germinational result of pecan pollens after 8 h rehydration. (A) and (D) no RT stored pollens. (B) and (E) RT-stored pollens for 3d. (C) and (F) RT-stored pollens for 7d. All pictures share same data tabs indicated in A.
restricted in space on well-germinated situation than on poorgerminated situation. Thus if the pollen tube length is used to evaluate the pollen viability, the pollen density should be adjusted to balance the germination rate and pollen tube growth, which is easily achieved in our method by adjusting the concentration of pollen suspension. In addition, we here tried a new parameter GT50 to indicate the speed of pollen germination, which is in fact the relative velocity of germination rate and germinational efficiency. This parameter revealed some information that germination rate and pollen tube length can’t provide including the influence of RT storage on germination speed. The analysis of data from GT50 indicated RT storage can obviously increase the germination time at low germination temperature although the final germination rate is less inhibited. Thus the GT50 can be taken as the early indicator of pollen physiological change during RT storage. 4.3. The influence of environmental variance on pollen germination Pollen germination is unique and important to flowering plant development, which emerges as a highly dynamic and co-ordinated process, integrating many different signals from the local environment (Taylor and Hepler, 1997). Due to the low metabolic activity of the pollen grains caused by its dehydrated state especially in desiccation-tolerant pollens (Edlund et al., 2004), the pollen grains must first rehydrate before it can restart metabolic processes and then germinate. In nature, the two processes, rehydration and germination, which desiccated pollen grains undergo usually occur
on same stigma, which are regulated both temporally and spatially. For pecan, pollen becomes rounded and hydrated by 1 hr after pollination and pollen tube emergence is visible within 3 h (Wetzstein, 1989). But our experiments proved that pollen can fulfill rehydration best in comparatively low temperature at which germination can’t proceed. And this temperature no doubt lies in the night time of pollination period in nature, suggesting the rehydration and germination process of pollen can be entirely separated even in nature. Although pollen usually begins shedding in mid to late morning, the pollen grains from wind-pollinated plants are small and light and can stay suspended in air for a long time (from hours to days) especially in hot, dry and windy days. Based on these phenomena together with our trials in catching pecan pollen grains with plate in field, we think that it is completely probable for the suspended pecan pollen from one tree to land on stigmas of the other tree much more later than pollen shedding. These situations provide some contacts for our in vitro simulation of the influence of environmental variance on pollen germination. In fact, temperature variance between day and night may generally have important influence on plant reproductive process including pollination and fruit ripe. For example, in tomato, although the number of abnormal pollen under high day temperature/low night temperature increases, this is more than compensated by an increased number of normal pollen and increased germination potential, and doesn’t reduce the pollen production and pollen germination potential (Khanal et al., 2013). And this paper may reveal some mechanism related to hydration on the effect of high day temperature/low night temperature in plant reproductive process.
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Fig. 7. The geminational results of newly thawed pecan pollens under different cold treatment after 4 h rehydration at 22 ◦ C. (A)–(C), results from cold treatment for 4 h. (D)–(F), results from cold treatment for 8 h. All pictures share same data tabs indicated in C.
5. Conclusions We’ve developed a mixing-spreading based method for pecan pollen germination in vitro which provide unprecedented level of the germinational repeatability and observability. Using this method, we found the existence of optimal combination of 16 ◦ C rehydration temperature and 25 ◦ C germination temperature, indicating the promotion of proper low rehydration temperature to the germination of pecan pollen in vitro. Combined with the data from germination rate, pollen tube length and germination speed, we discovered that pecan pollen didn’t lose its germinational ability rapidly in first several days but changed its optimal rehydration and germination condition obviously. In addition, we proposed here a new germination speed parameter GT50 to indicate the early change of pollen viability. Acknowledgments This work was supported by The State Bureau of Forestry 948 project (Grant no. 2011-4-31 to T.J.Z) and Zhejiang Provincial Natural Science Foundation of China (Grant no. Y3110499 to T.J.Z, grant no. Y3110530 to Q.Y.J and grant no. LY12C16001). References Conner, P.J., 2011. Optimization of in vitro pecan pollen germination. Hortscience 46 (4), 571–576. Crowe, J.H., Hoekstra, F.A., Crowe, L.M., 1989. Membrane phase transitions are responsible for imbibitional damage in dry pollen. Proc. Natl. Acad. Sci. U.S.A. 86 (2), 520–523. Edlund, A.F., Swanson, R., Preuss, D., 2004. Pollen and stigma structure and function: the role of diversity in pollination. Plant Cell 16 (Suppl.), S84–S97.
Galleta, G., 1983. Pollen and seed management. In: Moore, J., Janick, J. (Eds.), Methods in Fruit Breeding. Purdue University Press, West Lafayette, IN, pp. 23–47. Holdaway-Clarke, T.L., Weddle, N.M., Kim, S., Robi, A., Parris, C., Kunkel, J.G., Hepler, P.K., 2003. Effect of extracellular calcium, pH and borate on growth oscillations in Lilium formosanum pollen tubes. J. Exp. Bot. 54 (380), 65–72. Khanal, B., Suthaparan, A., Hückstädt, A., Wold, A., Mortensen, L., Gislerød, H., 2013. The effect of high day and low night temperature on pollen production, pollen germination and postharvest quality of tomatoes. Am. J. Plant Sci. 4 (7A), 19–25. Peng, F.R., Li, Y.R., Hao, M.Z., Liang, Y.W., Tang, P.P., Zhai, M., 2012. The present situation and development strategy of pecan in China. China For. Sci. Technol. 26 (4), 1–4. Rodriguez-Enriquez, M.J., Mehdi, S., Dickinson, H.G., Grant-Downton, R.T., 2013. A novel method for efficient in vitro germination and tube growth of Arabidopsis thaliana pollen. New Phytol. 197 (2), 668–679. Sparks, D., Madden, G., 1985. Pistillate flower and fruit abortion as affected by cultivar, time, and pollination. J. Am. Soc. Hortic. Sci. 110, 219–223. Sparks, D., Yates, I.E., 2002. Pecan pollen stored over a decade retains viability. Hortscience 37 (1), 176–177. Taylor, L.P., Hepler, P.K., 1997. Pollen germination and tube growth. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 461–491. Wetzstein, H., Rodriguez, A.M., Burns, J.A., Magner, H., 1996. Carya illinoensis (Pecan). 35, 50–75. Wetzstein, H., Sparks, D., 1985. Structure and in vitro germination of the pollen of pecan. J. Am. Soc. Hortic. Sci. 110, 778–781. Wetzstein, H.Y., 1989. Pollination and development of the receptive stigma in pecan, carya illinoensis. ISHS Acta Hortic. 240, 193–196. Wetzstein, H.Y., Sparks, D., 1986. Flowering in Pecan. Horticultural Reviews, 8. John Wiley & Sons, Inc., pp. 217–255, http://dx.doi.org/10.1002/9781118060810.ch6. Yates, I., Thompson, T., Giles, J., 1986. Proper pollen storage, germination tests essential to success of artificial pollination. Pecan South 20, 23–27. Yates, I.E., Sparks, D., Connor, K., Towill, L., 1991. Reducing pollen moisture simplifies long-term storage of pecan pollen. J. Am. Soc. Hortic. Sci. 116 (3), 430–434. Yates, I.E., Sparks, D., 1989. Hydration and temperature influence in vitro germination of pecan pollen. J. Am. Soc. Hortic. Sci. 114 (4), 559–605. Zhang, R., Li, H., Peng, F., Hao, M., Zhai, M., 2014. Flowering morphological characteristics, pollen viability and stigma receptivity of Carya illinoinensis. J. Nanjing Forestry University (Nat. Sci. Ed.) 38 (3), 50–54. Zhang, R., Li, Y., Liang, Y., Peng, F., Li, Y., 2013. In vitro pollen germination and tube growth characteristics in pecan (Carya illinoinensis). Acta Bot. BorealiOccidental. Sin. 33 (9), 1916–1922.
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A novel in vitro germination method revealed the influence of environmental variance on the pecan pollen viability Hua-Zheng Peng 1 , Qun-Ying Jin 1 , Hua-Lin Ye, Tang-Jun Zhu ∗ Zhejiang Provincial Key Lab of Bamboo Research, Zhejiang Forestry Academy, Hangzhou 310023, China
a r t i c l e
i n f o
Article history: Received 25 August 2014 Received in revised form 10 October 2014 Accepted 30 October 2014 Keywords: Pecan pollen Pollen germination Pollen rehydration Pollen tube growth Pollen viability
a b s t r a c t Pecan is one of the most important horticultural nut crops in the world. Germination tests are vital to the safe application of artificial pollination and cross breeding of pecan. Although many in vitro germination methods have been proposed, the realization of robust and reproducible methods to induce morphologically normal growth pollen tubes in vitro for detailed dynamic observation has often proved challenging in pecan. We employed a mixing and spreading method to realize the uniform distribution and germination on agarose medium, which resulted in the unprecedented level of germinational repeatability and observability. With this method, we investigated the influence of environmental variance on the in vitro germination of pecan pollen. The data from germination rate, burst rate, pollen tube length and germination speed indicated the pecan pollen had an optimal germination temperature at 25 ◦ C but optimal rehydration temperature at 16 ◦ C which was lower than minimum germination temperature. And this optimal rehydration temperature was harmful to pollen germination if pollen was ready to germinate after rehydration. It helped researchers understand the mechanism of probable promotion of high day/low night temperatures to pollen germination in nature. Results further revealed that pecan pollen didn’t lose their viability rapidly in first several days at ambient conditions but changed to a different rehydration and germination condition. In addition, we proposed here a new germination speed parameter GT50 to indicate the early change of pollen viability. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Pecan (Carya illinoinensis), belonging to the Juglandaceae family, originally grows in south-central North America and is one of the most important horticultural nut crops in the world (Wetzstein et al., 1996). It is a wind-pollinated monoecious plant with staminate flowers organized into an ament or catkin and female flowers borne on a spike (Wetzstein and Sparks, 1986). Because of the heterodichogamy and short flowering period, breeding programs often have to use stored pollen to perform particular pecan crossing. Efficient and reproducible viability testing before crossing is essential to avoid the use of nonviable stored pollen, or it will result in the loss of the cross for the year (Sparks and Madden, 1985). Germination tests have generally been considered to be the best in vitro indicator of pollen usefulness, which assess the viability of pollen samples by germinating a sample of pollen grains in an artificial media (Galleta, 1983). Previous studies have proved that the in vitro
∗ Corresponding author. Tel.: +86 571 87798231; fax: +86 571 87798113. E-mail address: [email protected] (T.-J. Zhu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.scienta.2014.10.057 0304-4238/© 2014 Elsevier B.V. All rights reserved.
germination of most pollens are mainly affected by the osmotic potential regulator (such as sucrose), concentration of borate and so on in medium although the effects of these ingredients in medium are not completely understood (Holdaway-Clarke et al., 2003). In pecan, trials have proved that fresh pollen can germinate in an artificial medium (Wetzstein and Sparks, 1985; Yates et al., 1986) and controlled rehydration of dry pollen before placing in the germination media is vital to determining accurate germination rate (Yates and Sparks, 1989). The rehydration experiment of dry pecan pollen helps prove that pollen stored for nearly 2 years at −80 ◦ C or −196 ◦ C, but not −10 ◦ C, can retain germination capacity equal to freshly collected pollen by both in vitro germination test and fruit set analysis (Yates and Sparks, 1989). Further studies reveal that pecan pollen maintained at −12 ◦ C for 2 years is as viable as freshly collected pollen if it is oven dried at 35 ◦ C to a constant weight and stored in moisture-proof bags (Yates et al., 1991). Some experiments even reveal that the viability of pecan pollen does not decline in frozen storage up to 13 years (Sparks and Yates, 2002). On the other hand, researchers also find that pecan pollen loses their viability rapidly in first several days although it can retained the low germinational capacity for 59 days at ambient laboratory conditions (Yates and Sparks, 1989). It is surprising that pecan pollen will
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lose its viability rapidly in short time since this type of desiccationtolerant pollen can survive for a long period of time after anthesis. In addition, when it is introduced from its original region into other climate zone, pecan may encounter some problems in pollination and germination although the seedlings still grow well (Peng et al., 2012). To deeply explore the detailed influence of environmental variance on pollen viability, we need a high robust and reproductive method to induce in vitro pollen germination and morphologically normal growth of pollen tube. But the designing of such method in pecan is always a challenging. In recent years, a new cellophane based method is proposed to improve the germination rate and repeatability of pecan pollen, which has considerable utility compared to the traditional drop, tube, multi-well liquid and multi-well agar methods (Conner, 2011). However, there still exist some obvious defects in current method. First, non-replicable results still exist due to the use of ‘dusting’ method to spread pollen on medium surface. Pollen density can influence the germination of plant pollen no exception of pecan pollen whose germination proves decreasing below or above the optimal concentration (Yates et al., 1986). Second, the current method can’t be used to observe the germination process directly and dynamically. The advancing of novel method in model plant Arabidopsis thaliana using agarose and cellophane as support greatly facilitates efficient, reproducible studies of the post-hydration development of pollen from A. thaliana and its relatives (Rodriguez-Enriquez et al., 2013). But, for in vitro germination of pollen from pecan and its relatives, a robust and reliable germination method, through which germination process of differently treated pollen can be compared in detail, has remained elusive. In this paper, we proposed a novel method for efficient in vitro germination with very high repeatability which can not only promote germination and tube growth, but also realize dynamic observation of germination process. We used this system to evaluate the influence of environmental variance on in vitro germination of pecan pollen. 2. Materials and methods 2.1. Collection and use of pollen Studies on the pollen germination were conducted using ‘Pawnee’ pollen collected in early May of 2013. Pollens were collected, dried and stored according to the reference (Conner, 2011). They were aliquoted into 50-ml polypropylene centrifuge tubes and frozen at −80 ◦ C until use. Germination tests were conducted using newly thawed and RT-stored pollen samples. For a newly thawed pollen sample, a 50-ml sample tube was taken out of the freezer and immersed in ice to thaw for 2 h and then transferred to dessicator at room temperature for 2 h. For RT-stored pollen sample, the above-mentioned sample tube was finally stored at dessicator at room temperature for 3d or 7d before germination tests. 2.2. Pollen culture medium and apparatus Germination medium was mainly made on the base of previous studies with some necessary optimization for the method in this paper (Conner, 2011). It consisted of 20% sucrose, 4 mM Boric acid, 3 mM CaCl2 , 0.67 mM KCl, 10 mM MES-Tris (pH5.8) and 1% agarose (Biowest, Spain). The liquid part of the germination medium was made by dissolving sucrose first and then adding the other materials except agarose. The liquid part was used here to mix rehydrated pollen for spreading. For the final germination medium, the appropriate agarose was added to the above solution and briefly heated in a microwave. Each 5 ml medium was then poured into 5 cm petri dish before its solidification and blown openly in clean bench for
10 min. The plate can be stored at 4 ◦ C temporarily for a couple of days before use. 2.3. Pollen germination method Germination tests here included several necessary steps (Fig. 1A). First, the thawed pollen was rehydrated for hours before transferring to germination medium. In this step, pollen samples were dusted into petri dishes and suspended above water in a sealed container at required rehydration temperature. Second, the rehydrated pollen was weighed into 1.5 ml centrifuge tube and then fully mixed with liquid germination medium in proportion by tip. Here 10 l suspension was dropped into the germination medium plate for a single test right after the mixing. Third, the pollen suspension was spread evenly on plate using cell spreader (1 cm side length) by a unidirectional spreading. Each germination plate was immediately covered after spreading to prevent drying. Finally, the plate, suspended above water in a sealed box, was incubated in the dark at certain temperature to germinate for at least 24 h. When taken for examination every 3 h, the plate was covered and observed directly by inverted microscope. Each treatment of every sample was repeated three times. 2.4. Microscopy and data analysis During the germinational process, the germinational phenomena in all tests were observed and recorded by Olympus inverted fluorescence IX71 microscope with DP71 camera at 100× magnification. A pollen grain was considered to have germinated if the pollen tube was equal to or greater in length than the width of the pollen grain. And a pollen grain was considered to be burst if the inclusion of pollen diffuse. The grain both have germination and burst (usually burst from the pollen tube) will be considered as germination instead of burst. Germination images were further measured including count and pollen tube measurement by Image Pro Plus 6.0. In each replication, three vision fields were examined with each field containing 200 or so pollen grains. The data were exported to Excel software for further analysis including arithmetic mean and error analysis. The arithmetic mean of data from three vision fields was taken as one experimental repeat. For length measurement of pollen tube, 50 pollen tube or all (if the quantity was not enough) was measured from each of three vision fields and their arithmetic mean was taken as one experimental repeat. Experimental errors were estimated through data from three repetition. Paired t-test was performed in Excel for group data comparison and all indicated P values were two tailed. 3. Results 3.1. The selection strategy of rehydration and germination condition We used ultra cold stored pollen samples instead of freshly collected pollen samples in our experiments, which is on one hand because of better repeatability and convenience, on the other hand because of equal germination capacity to freshly collected pollen by both in vitro germination test and fruit set analysis (Sparks and Yates, 2002; Yates et al., 1991; Yates and Sparks, 1989). In our preliminary experiments, we used the mixing and spreading method to make sure the optimal pollen concentration for high germination rate, which was about 0.02 g rehydrated pollen per 100 l liquid germination medium for the sample we used. Our studies also indicated that the pollen could hardly germinate below 22 ◦ C which was then set as the temperature boundary between rehydration and germination. We also noticed from the climate record in pecan’s naive region that the day mean minimum temperature
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Fig. 1. Flow chart and corresponding effect of the novel germination method of pecan pollen in vitro. (A) The flow chart of germination method. The solid line stands for the real operation in this paper while the dotted line indicates the future use. (B) Pecan pollens spread in 5 cm Petri dish. (C) Germination observation of pecan pollen in the abovementioned B under inverted microscope. The white arrows indicate burst. (D) Germination result of pecan pollen using cellophane-agarose method (Rodriguez-Enriquez et al., 2013). Bar = 100 m.
was 10 ◦ C or so and the day mean maximum temperature was about 31 ◦ C during the flowering season. Then the scope of rehydration temperature was set from 4 ◦ C to 22 ◦ C while the scope of germination temperature was set from 22 ◦ C to 31 ◦ C. The 4 ◦ C was chosen for the reason that this temperature is widely used in the cold treatment and temporary storage of biology sample. For rehydration time, 4 h was selected as standard while the double time (8 h) was chosen as prolonged time according to the references and our preliminary experiments (Conner, 2011). The effect of cellophane method which has been successfully used in A. thanliana (Rodriguez-Enriquez et al., 2013) was compared to the present method, indicating the obviously better uniformity and visuality of method proposed here (Fig. 1).
3.2. The proper low rehydration temperature promoted pollen germination The newly thawed pecan pollen was used to estimate the influence of rehydration temperature and germination temperature on germination. The data indicated that germination rate rose with the increase of rehydration temperature only when the rehydration temperature was below 16 ◦ C no matter how long the rehydration time was (Fig. 2A and C). The comparison of
germination results between 16 ◦ C and 22 ◦ C rehydration temperature could be further analyzed by statistical method (Table 1), indicating the significant improvement at 16 ◦ C rehydration condition for no RT-stored pollen samples. And the germination rate rose with the increase of germination temperature only when the germination temperature was below 25 ◦ C. While the pollen burst rate mainly rose with the increase of germination temperature from 22 ◦ C to 31 ◦ C and less affected by the rehydration temperature (Fig. 2B and D). Thus the best germination result with the highest germination rate and comparatively low burst rate occurred at a combination of 16 ◦ C rehydration temperature and 25 ◦ C germination temperature under both standard and prolonged rehydration time. The results definitely showed that the rehydration temperature was comparably important to pecan pollen germination with germination temperature. And the rehydration temperature lower than minimum germinational temperature was beneficial to pecan pollen germination.
3.3. RT storage first changed pollens’ optimal rehydration condition We then investigated the germination results of RT-stored pecan pollens. After being stored at RT for 3d and 7d, the pollen
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Fig. 2. The germinational result of newly thawed (no RT-stored) pecan pollen under different rehydration and germination conditions. (A) The germination rate after 4 h rehydration. (B) The burst rate after 4 h rehydration. (C) The germination rate after 8 h rehydration. (D) The burst rate after 8 h rehydration. All pictures share same data tabs indicated in B.
Table 1 Comparison of germination rates between rehydration temperature 16 ◦ C and 22 ◦ C by statistical test. Pollen sample no RT-stored pollen
Rehydration Temp ( C)
Temp ( C)
Rate (%)
16
22 25 28 31 22 25 28 31 22 25 28 31 22 25 28 31
36.0 50.6 40.1 36.2 30.9 48.6 38.7 33.9 23.3 41.8 35.3 33.3 21.8 37.4 34.1 29.9
0.047*
22 25 28 31 22 25 28 31
17.6 31.3 27.7 25.4 11.2 19.8 25.0 22.3
0.062
16
22
4
16
22
P < 0.05.
P value
4
8
*
◦
Time (h)
22
3d RT-stored sample
Germination ◦
0.043*
samples under standard rehydration time showed similar germination pattern including the appearance of optimal rehydration temperature (P = 0.062, Table 1.) except for the lower germination rates (Figs. 3A and 4A). But it was surprising that under the prolonged rehydration time the germination rate of pollens increased rapidly at rehydration temperature 22 ◦ C as if the ‘sleeping’ viability had been reactivated (Figs. 3C and 4C). And the germinate rate of RT-stored sample was so high that it could reach more than 85% of the highest level of no RT-stored samples when the RTstored sample was reactivated by higher rehydration temperature and prolonged rehydration time (Fig. 3C). On the other side, it was predictable that the burst rate increased with the increase of germination temperature and RT-stored time, especially germination at higher temperature such as 28 ◦ C and 31 ◦ C after longer RT-storage (Figs. 3B and D and 4B and D). Thus we deduced that the pecan pollens mainly changed their rehydration condition instead of losing their viability rapidly during the early days. 3.4. RT storage also changed pollen’s optimal germination condition Germinational data indicated that the optimal germination temperature was 25 ◦ C in no RT-stored and 3d RT-stored pollen samples (Figs. 2A and C and 3A and C), but it slowly changed to 28 ◦ C in 7d RT-stored sample (Fig. 4A and C). And it should be noticed that this optimal germination condition which led to the highest germination rate also led to the higher burst rate (Fig. 4B and D).
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Fig. 3. The germinational result of RT-stored (3d) pecan pollen under different rehydration and germination conditions. (A) The germination rate after 4 h rehydration. (B) The burst rate after 4 h rehydration. (C) The germination rate after 8 h rehydration. (D) The burst rate after 8 h rehydration. All pictures share same data tabs indicated in B.
This situation was further supported by the evidence from pollen tube length which is one of important indicators of pollen viability (Fig. 5). For no RT-stored pollen, the pollen tube lengths were close under almost all rehydration and temperature condition except 8 h rehydration at 22 ◦ C (Fig. 5A and D). For 3d RT-stored group, the difference of pollen tube lengths from different samples became large (Fig. 5B and E). And for 7d RT-stored group, 28 ◦ C had obviously become the optimal germination temperature based on average pollen tube length (Fig. 5C and F). In addition, it was found that too low germination temperature like 22 ◦ C after prolonged rehydration time was harmful to pollen tube growth (Fig. 5D and E).
25 ◦ C but not obviously at high temperature like 28 ◦ C and 31 ◦ C (Fig. 6A–C). The situation was quite different to prolonged rehydration time under which RT-storage can’t obviously increase GT50 of pollen sample (Fig. 6A–C). In addition, it was surprising that RT storage could sharply increase the GT50 of pollen samples rehydrated at 4 ◦ C although it usually less affected the GT50 of pollen samples germinated at high temperature (Fig. 6C). Thus, the evidence from germination time further indicated that the optimal rehydration and germination temperature tended to be higher temperature since the germination ability of pollen samples at lower temperature was more likely to be inhibited during RT storage.
3.5. Germination time also suggested the change of optimal germination temperature in RT-stored pollen
3.6. Cold treatment after rehydration impairs the pollen germination
Besides germination rate and pollen tube length, germination speed is another important parameter to evaluate the pollen germination ability. We here used the median germination time (GT50) as a substitute of germination speed which was defined as the time to reach half of the highest germination rate. The results indicated that pollen GT50 was mainly affected by rehydration time and germination temperature instead of rehydration temperature (Fig. 6). The data showed that higher germination temperature usually led to lower germination time, that was faster germination speed. Under standard rehydration time, RT storage would increase GT50 and the incremental degree depended on germination temperature, that was to say, RT storage can greatly increase GT50 of pollen sample germinated at low temperature like 22 ◦ C and
We have proved proper low rehydration temperature can promote the germination of pecan pollen in vitro (Fig. 2). But if the cold treatment occurs after standard rehydration, what the influence will be? Our data indicated that cold treatment was harmful to germination once the pollen had fulfilled its rehydration except 4 ◦ C treatment (Fig. 7A and D). And the optimal rehydration temperature above-mentioned, that was 16 ◦ C, on the contrary inhibited the pollen germination most. Besides, the low germination rates from cold treatments were accompanied by higher pollen burst rate compared to the germination without cold treatment (Fig. 7B and E). Only the pollen tube length seemed not to be suppressed by cold treatment (Fig. 7C and F). It is understandable that 4 ◦ C treatment didn’t restrict the pollen germination since this temperature
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Fig. 4. The germinational result of RT-stored (7d) pecan pollen under different rehydration and germination conditions. (A) The germination rate after 4 h rehydration. (B) The burst rate after 4 h rehydration. (C) The germination rate after 8 h rehydration. (D) The burst rate after 8 h rehydration. All pictures share same data tabs indicated in B.
can keep the pollen viability temporarily. But the influence of other cold treatments on rehydrated pollen was indeed surprising and it suggested that the low temperature exerts its promotional function only at early stage of rehydration.
4. Discussion 4.1. Advantages of this germination method of pollen in vitro The uniqueness, simplicity and independence of pollen germination as well as its importance to scientific and applied studies of plant reproduction, have created a strong demand for in vitro systems by which pollen germination and tube growth can be induced. Although numerous germination methods have been proposed before, a novel method mainly using cellulose membrane and spermidine is especially attractive, which is applied successfully in the germination and tube growth of A. thaliana pollen in vitro (Rodriguez-Enriquez et al., 2013). But when the aforementioned method in A. thaliana was used in the germination of pecan-like pollens, the observational effect was unacceptable mainly due to the serious uniformity problem and density-dependent pollen germination. In pecan, our own trials found that single liquid method also failed to exhibit the ideal germinational effect although it can distribute pollen grains uniformly in initial mixing step. This is obviously aggravated after pollen tube growth due to the twine of pollen tube, leading to the poor observability (Zhang et al., 2013, 2014).
In this paper, the liquid germination medium was used to thoroughly mix the pollen grains and cell spreader was used to disperse the pollen evenly on the medium surface, which are the two key elements of our method. Although it was very simple, it performed effectively and fully made use of the superiority of pollen system (a well-distributed cell suspension). Compared to the brushing or dusting methods used before, the mixing-spreading method led to much higher uniformity and greatly improved the repetitiveness and reliability of pollen experiments, which proved helpful in elucidating the influence of environmental factors on pollen germination in this paper. The mixing-spreading method also has good compatibility with previous methods, that is to say, if it’s needed, the pollen can also be spread on the cellophane surface or slide surface. And the successive cell biology studies such as dynamic observation and the location of sperm cells can also be performed in the method. It excludes an unnecessary worry that immersing pollen in liquid culture will inhibit its germination ability since this method only involves a transient mixture of liquid medium unlike the traditional liquid germination method. Furthermore, the small amount of liquid spreading with pollen will soon be absorbed by agarose medium since the solid medium has been blown to relatively dry state. In addition, the pollen will not sink into the medium and pollen tube almost can’t grow into the medium since the agarose concentration is relatively higher (1% agarose) instead of 0.5% agarose in A. thaliana method. In other words, these improvements not only can’t restrict the ventilation of germinational environment, but also can lead to adequate touch and even dispersing of pollen on medium, which finally realize the
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Fig. 5. The pollen tube length of differently stored pecan pollens under different rehydration and germination conditions. (A)–(C) The germinational result of pecan pollens after 4 h rehydration. (D)–(F) The germinational result of pecan pollens after 8 h rehydration. (A) and (D) no RT stored pollens. (B) and (E) RT-stored pollens for 3d. (C) and (F) RT-stored pollens for 7d. All pictures share same data tabs indicated in C.
excellent uniformity and direct observation of pollen germination. Finally, as far as germination rate produced by different germination methods is concerned, we think that they can’t be strictly compared by the results from different papers since the pollen samples may come from different cultivars and have other different environmental conditions not mentioned by the authors. But we know that the liquid method has ever been compared with cellophane method, indicating the relatively low germination rate of liquid method (Conner, 2011), which was also supported by our trials with same pecan cultivar. In this paper, the maximum germination rate brought by our method were equivalent to the result from cellophane method (Fig. 1) and the result from previous report (Conner, 2011). Thus, we think that this new method will greatly mine the potential of pollen system in studies of plant reproductive mechanism and in setup of plant cell model. 4.2. The evaluation of the viability of desiccation-tolerant pollen Presently there exist several methods to evaluate the pollen viability including viability stain and in vitro germination, among which the in vitro germination method is thought to be more persuasive (Galleta, 1983). A fixed so-called “optimal” condition is usually used in the determination and comparison of pollen viability. Through our experiments, we found in pecan the rehydration condition was one of the keys to understand the durable ability of pollen germination. That is to say, pecan pollen actually doesn’t lose their viability rapidly but move to a different state in first several days. If we continue using the initial experimental condition
including the rehydration condition to test pollen viability, the germination result is much worse than the real value. And this also reminds us of cautious using of in vitro germination method to compare pollen viability. In plants, it has proved that controlled rehydration allows the phospholipids in the plasma membrane bilayer to go through phase transition before being exposed to bulk water (Crowe et al., 1989). Thus the change of rehydration condition probably suggest the transition of physiological state of plasma membrane which is the mechanism of early change of pollen viability. There are also some other important parameters such as pollen tube length and germination speed to evaluate the pollen viability besides the germination rate during in vitro germination test. Pollen tube length is seldom mentioned in previous studies of pecan pollen probably due to the use of unstable method although it is widely used in many other species as one of important indicators to determine pollen germination. Based on this new germination method, we found pecan pollen tube length was quite different from the germination rate on the sensitivity to temperature. While the germination rate is obviously influenced by both rehydration and germination temperatures, the pollen tube length is mainly influenced by germination temperature which was obvious for RT-storage samples (Fig. 5B, C, E and F). And this effect of germination temperature on pollen tube length was not suppressed by burst since high germination rate at high temperature sometimes was accompanied by high burst (Figs. 4B and D and 5C and F). But there are also some deficiency in the current germination process when taking the pollen tube length as an indicator of pollen viability since the growth of pollen tube seemed more
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Fig. 6. The median germination time of differently stored pecan pollens under different rehydration and germination conditions. (A)–(C) The germinational result of pecan pollens after 4 h rehydration. (D)–(F) The germinational result of pecan pollens after 8 h rehydration. (A) and (D) no RT stored pollens. (B) and (E) RT-stored pollens for 3d. (C) and (F) RT-stored pollens for 7d. All pictures share same data tabs indicated in A.
restricted in space on well-germinated situation than on poorgerminated situation. Thus if the pollen tube length is used to evaluate the pollen viability, the pollen density should be adjusted to balance the germination rate and pollen tube growth, which is easily achieved in our method by adjusting the concentration of pollen suspension. In addition, we here tried a new parameter GT50 to indicate the speed of pollen germination, which is in fact the relative velocity of germination rate and germinational efficiency. This parameter revealed some information that germination rate and pollen tube length can’t provide including the influence of RT storage on germination speed. The analysis of data from GT50 indicated RT storage can obviously increase the germination time at low germination temperature although the final germination rate is less inhibited. Thus the GT50 can be taken as the early indicator of pollen physiological change during RT storage. 4.3. The influence of environmental variance on pollen germination Pollen germination is unique and important to flowering plant development, which emerges as a highly dynamic and co-ordinated process, integrating many different signals from the local environment (Taylor and Hepler, 1997). Due to the low metabolic activity of the pollen grains caused by its dehydrated state especially in desiccation-tolerant pollens (Edlund et al., 2004), the pollen grains must first rehydrate before it can restart metabolic processes and then germinate. In nature, the two processes, rehydration and germination, which desiccated pollen grains undergo usually occur
on same stigma, which are regulated both temporally and spatially. For pecan, pollen becomes rounded and hydrated by 1 hr after pollination and pollen tube emergence is visible within 3 h (Wetzstein, 1989). But our experiments proved that pollen can fulfill rehydration best in comparatively low temperature at which germination can’t proceed. And this temperature no doubt lies in the night time of pollination period in nature, suggesting the rehydration and germination process of pollen can be entirely separated even in nature. Although pollen usually begins shedding in mid to late morning, the pollen grains from wind-pollinated plants are small and light and can stay suspended in air for a long time (from hours to days) especially in hot, dry and windy days. Based on these phenomena together with our trials in catching pecan pollen grains with plate in field, we think that it is completely probable for the suspended pecan pollen from one tree to land on stigmas of the other tree much more later than pollen shedding. These situations provide some contacts for our in vitro simulation of the influence of environmental variance on pollen germination. In fact, temperature variance between day and night may generally have important influence on plant reproductive process including pollination and fruit ripe. For example, in tomato, although the number of abnormal pollen under high day temperature/low night temperature increases, this is more than compensated by an increased number of normal pollen and increased germination potential, and doesn’t reduce the pollen production and pollen germination potential (Khanal et al., 2013). And this paper may reveal some mechanism related to hydration on the effect of high day temperature/low night temperature in plant reproductive process.
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Fig. 7. The geminational results of newly thawed pecan pollens under different cold treatment after 4 h rehydration at 22 ◦ C. (A)–(C), results from cold treatment for 4 h. (D)–(F), results from cold treatment for 8 h. All pictures share same data tabs indicated in C.
5. Conclusions We’ve developed a mixing-spreading based method for pecan pollen germination in vitro which provide unprecedented level of the germinational repeatability and observability. Using this method, we found the existence of optimal combination of 16 ◦ C rehydration temperature and 25 ◦ C germination temperature, indicating the promotion of proper low rehydration temperature to the germination of pecan pollen in vitro. Combined with the data from germination rate, pollen tube length and germination speed, we discovered that pecan pollen didn’t lose its germinational ability rapidly in first several days but changed its optimal rehydration and germination condition obviously. In addition, we proposed here a new germination speed parameter GT50 to indicate the early change of pollen viability. Acknowledgments This work was supported by The State Bureau of Forestry 948 project (Grant no. 2011-4-31 to T.J.Z) and Zhejiang Provincial Natural Science Foundation of China (Grant no. Y3110499 to T.J.Z, grant no. Y3110530 to Q.Y.J and grant no. LY12C16001). References Conner, P.J., 2011. Optimization of in vitro pecan pollen germination. Hortscience 46 (4), 571–576. Crowe, J.H., Hoekstra, F.A., Crowe, L.M., 1989. Membrane phase transitions are responsible for imbibitional damage in dry pollen. Proc. Natl. Acad. Sci. U.S.A. 86 (2), 520–523. Edlund, A.F., Swanson, R., Preuss, D., 2004. Pollen and stigma structure and function: the role of diversity in pollination. Plant Cell 16 (Suppl.), S84–S97.
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