We examined the influence of storage time on germinability and tube growth of freeze stored pollens collected from 25 wild male plants in
Actinidia eriantha variety. Pollens were stored in freezer at - 20°C for six months and one year periods to determine changing at germinability in time.
In vitro germination was conducted in certain cultural medium defined for
Actinidia genus. The results showed that the germination percentages and tube lengths of genotypes decreased at the end of storage period. MH22, MH45, MH47, MH56, MH67, MH70, MH71, MH72, MH74, MH55 and MH61 genotypes were evaluated as vigor genotypes, because they maintained their viability and germination capability displaying statistically insignificant decreasing although their tube lengths significantly decreased except MH67. This investigation provided to determine some robust wild male germplasm resources in
A. eriantha in point of durability of pollens against long term conservation for using at future pollination and breeding programs.
Kiwifruit (genus Actinidia) is well known for its great medicinal importance and has been considered for various treatments and onsets [1] . The variety Actinidia eriantha Benth is a valuable breeding material, as the vitamin C content of the fruit is 1013.98 mg/100g fresh weight (f.wt.), which is much higher than that of A. deliciosa (200 - 370 mg/100g f.wt.) and A. chinensis (250 - 300 mg/100g f.wt.) [2] . Its fruit size is the third largest for variety in this genus [3] . Therefore, cultivated and wild germplasm resources of A. eriantha variety carry considerable importance to utilize in breeding and propagation studies for getting productive, high quality and new varieties. High quality and yielding at fruit production absolutely depend on good fertilization following successful pollination. In this regard, the pollens of A. eriantha variety as genetic source were also used at researches made on pollen morphological and physiological traits.
Recently the studies on pollen physiological traits especially germination and viability have received substantial attention for their application in plant breeding, conservation and so on [4] . Pollen has remarkable potential to achieve genetic transformation [5] . Long-term storage of pollen grains is a useful method for conservation of genetic factors to use in the breeding programs; on the other hand, storage of pollen is necessary for controlled pollination, getting desired characters by breeding program and to overcome complications emerging at cultural implementations [6] . Studies on pollen traits especially germination percentage and tube growth in stored pollens should be carried out for their viability and longevity in different researches and horticultural exercises [7] .
Effects of storage conditions on pollen viability have been reported by many researchers. For instance, according to an investigation about germination capacity of stored pollen of Solanum melongena L., maintaining the germination capacity of stored pollen can be useful in time saving in hybridization programs and also in crops improvement, the most important factors for successful pollen conservation are storage temperature and moisture content of material; lowering both tends to increase the period of viability so pollen stored in freezer (−30˚C, −20˚C) showed better germination percentage as compared to pollen stored at +4˚C and in organic solvents [4] . Pollens kept their viability high after 18 months of storage at −20˚C and also controlled pollination with pollens stored for 12 months resulted well in seed set which was comparable to that obtained using fresh pollen in Salix [8] . Another research on pear indicated that pollens from early blooming varieties can be stored for very long period without any appreciable loss of viability and germination so that pollen stored at low temperature (−120˚C and −20˚C) showed better viability and germination percentage as compared to pollen stored at room temperature and 4˚C [6] . Best results were obtained after 24 months at −20˚C in date palm pollens [9] . Pollen stored at low temperature (−30˚C, −20˚C) showed better germination as compared to pollen stored at 4˚C in the fresh Abelmoschus esculentus L. [5] .
Although low temperature storage is an effective way to maintain viability for lots of species, long time preservation can cause decreasing in viability and germinability for some species. Such as pollen stored in freezer at −20˚C and −30˚C showed good germination but progressive storing time caused the gradually decreasing at germination percentage in Pisum sativum L. [10] . The viability showed a decreasing trend with increase in storage period and thus an inverse relation between viability and duration of storage was observed in three pear cultivars Pathernakh, Punjab Beauty and Shinseiki [6] . Similar observations were also obtained from cherimoya [11] and potato pollens [12] .
Decreasing in pollen germination along long storage period can be derived from physiological or genetic causes. According to study related with Anigozanthos manglesii, year-long storage will probably require isolation of pollen because successful freezing requires rapid energy transfer between tissue and coolant, probably sufficiently to allow ice crystal formation and damage to the pollen [13] . Sometimes, cultivars produce high quantity of pollens but not with high quality such as low pollen germination percentage or low tube growth also, some of the pollens may be sterile or not viable [7] .
Few studies have been published on in vitro viability and storage conditions of pollen grains of kiwifruit [14] . It was observed in kiwifruit, after 365 days of storage, the pollen grains had completely lost the ability to germinate in culture medium so strategies for short term conservation of pollen grain may be required, although the viability may be reduced during storage of pollen grains, which could decrease the efficiency of pollination [15] .
In this study, we examined the influence of storage time on Pollen Germination (PG) and Pollen Tube Length (PTL) of freeze stored pollens collected from 25 wild male germplasm resources in Actinidia eriantha variety.
2. Material and Methods
The well-grown flower clusters were pruned off from twenty five wild male genotypes (MH10, MH22, MH26, MH30, MH31, MH34, MH41, MH43, MH45, MH46, MH47, MH48, MH55, MH56, MH57, MH58, MH60, MH61, MH66, MH67, MH69, MH70, MH71, MH72 and MH74) of A. eriantha variety growing at Magu Mountain region in Nancheng county, Fuzhou city, Jiangxi province, southeast of China (27˚25'N - 27˚32'N & 116˚27'E - 116˚32'E) at the beginning of flowering season in early May.
Flower clusters as experimental materials, which were immediately kept into cold preservation box after picked up in field, were transported to the laboratory of Agronomy Department, Jiangxi Agricultural University in Nanchang city. In the laboratory, Sepals and petals were separated and anthers were placed in the sterile labeled petri dishes. Anthers were kept in the laboratory at room temperature until dry in order to dehiscence and then pollens were collected into paper bags by vibrating anthers with traditional hand method. Collected pollens were stored in freezer at −20˚C for six months and one year periods to determine changing at pollen germinability in time.
In vitro germination assays determine the actual germination ability of pollen under suitable conditions [16] . Pollen germination in cultural medium is a useful viability test. Results of many researches showed that calcium, boric acid, sucrose, temperature and pH had important roles in germination tests for different species and varieties. Such as, for almond pollens it was suggested that addition of B to the culture media significantly increased pollen germination and pollen tube growth [17] . The effect of either sucrose or boric acid individually showed good results, but sucrose in combination with boric acid promoted pollen germination as well as tube development, because boron makes a complex with sugar and this sugar-borate complex is known to be capable of better translocation than non-borate, non-ionized sugar molecules [18] . Calcium concentration also was important for pollen germination, but more critical for normal pollen tube growth and to consolidate the view that temperature was the critical factor determining reproducibility of in vitro pollen germination [19] . The role of calcium and boric acid were observed at stored almond and peach pollen [20] . The importance of boric acid, calcium, temperature and sucrose in culture medium was also declared for almond pollen [21] . The role of pH was investigated in banana [22] .
The most suitable culture medium for kiwifruit pollen germination and tube growth was 10% sucrose + 100 mg∙L−1 boric acid + 10 mg∙L−1 Ca(NO3)2. The suitable culture time was 5 hours, the best temperature for pollen germination and tube growth was 30˚C and the suitable pH value of culture medium was 6 [23] . 8% agar was added into liquid culture medium including distilled water to solidify. Culture medium was heated in the microwave oven. A few drops hot culture medium were added on the microscope slides. Two slides were used for per genotypes. The pollens were gently dusted on the solid culture medium placed on slides using hair brash. The labeled microscope slides were put into the sterile petri dishes with moistened filter paper and incubated in dark. Pollen germination was observed under a light microscope (40 × 10) with Image-Pro Plus 6.0 software. Pollen was considered to have germinated when the length of the pollen tube was equal to or longer than the diameter of the pollen. Germination data were obtained on a sample of 100 pollen grains with 4 replications (four view points in two slides) by Analyzer software program. Mean pollen tube length of each genotype was recorded by averaging of 40 pollen grains using Tsview software program (10 mm = 100.000 photo pixel).
Statistical analysis were carried out using Microsoft Excel (2010), SPSS version 20 software with one way ANOVA and MANOVA test methods. Means were compared using Tukey test (p ≤ 0.05).
3. Results and Discussion
Pollen grains, being the sexual reproductive unit and the carrier of male genetic material in higher plants, play a vital role in breeding programme and assist successful fruit-set. High crop yield generally depends on viable pollen grains. Pollen fertility and viability have a paramount importance in hybridization programme. Pollen performance in terms of germination ability may have the relative importance with fruit-set [24] . In this research, main pollen traits including germination, tube growth and longevity of 25 wild male plants in A. eriantha were examined to determine germinability at two different periods of time intervals as six months and one year in freeze storage (−20˚C). It was said in the research of in vitro germination and pollen conservation of some Musa species that wild plants are often considered as genes bank and useful to solve problems in cultivated species [25] . Some other works were carried out by several researchers such as in Arabidopsis thaliana [26] and Morus alba [27] .
In the first experiment, mean pollen germination percentages (%/˚C) and tube lengths (10 mm = 100.000 photo pixel/˚C) of 25 wild male genotypes in A. eriantha were compared after six months storage by variance analysis (Table 1). The genotypes were separated into nine different statistical groups (a, b, c, d, e, f, g, h, i) in the pollen germination percentages according to variance analysis. Pollen germination was ranged between 25% and 91.50%. As result, MH48 (25%) and MH67 (26.25%) were in the lowest group (a). MH74, MH57 and MH30 were in the second group (ab) with low values. MH66 alone had highest germination percentage in the last group (i). Pollen germination percentages of MH58, MH10, MH43, MH41, MH69, MH70, and MH71 were
Comparison of mean pollen germination percentages (%/˚C) and tube lengths (10 mm = 100.000 photo pixel/˚C) of 25 wild male genotypes in A. eriantha after six months storage
Germination percentage
Pollen tube length
Genotype
N1
Result
Genotype
N2
Result
MH48
4
25.00a
MH74
40
26.22a
MH67
4
26.25a
MH57
40
26.53a
MH74
4
37.75ab
MH34
40
32.05ab
MH57
4
39.00abc
MH48
40
32.17ab
MH30
4
44.75abcd
MH72
40
32.66ab
MH26
4
51.00bcde
MH47
40
32.77ab
MH47
4
51.50bcde
MH45
40
35.23abc
MH56
4
52.50bcde
MH67
40
35.46abc
MH31
4
55.00bcde
MH26
40
38.48abcd
MH60
4
56.25bcdef
MH56
40
40.00abcd
MH34
4
56.75bcdef
MH41
40
40.09abcd
MH72
4
56.75bcdef
MH10
40
43.54bcd
MH22
4
57.00bcdef
MH61
40
44.34bcd
MH46
4
57.75bcdef
MH55
40
45.51bcd
MH61
4
62.75cdefg
MH46
40
46.36bcd
MH55
4
65.75defg
MH22
40
48.07cd
MH45
4
68.00defgh
MH60
40
48.44cd
MH71
4
69.25defghi
MH71
40
51.09de
MH70
4
69.75defghi
MH69
40
53.04def
MH69
4
73.00efghi
MH70
40
62.52efg
MH41
4
74.50efghi
MH31
40
65.77fg
MH43
4
79.50fghi
MH30
40
68.08g
MH10
4
82.75ghi
MH43
40
81.30h
MH58
4
89.50hi
MH66
40
86.52h
MH66
4
91.50i
MH58
40
88.95h
“N1” expresses total replication counts; “N2” expresses total pollen grain counts.
ranged between 89.5% and 69.25% following MH66. On the other hand, the genotypes were separated into eight statistical groups in pollen tube length. The tube length was ranged between 26.22 mm (MH74) and 88.95 mm (MH58). MH57, MH34, MH48, MH72 and MH47 showed low values in two groups “a” and “b” following MH74. MH43 (86.52 mm) and MH66 (81.30 mm) displayed highest values in same group with MH58.
In the second experiment, mean pollen germination percentages and tube lengths of genotypes were compared by variance analysis after one year storage (Table 2). MH10, MH30, MH31, MH34, MH43, MH57, MH58 and MH66 completely lost their viability at the end of one year storage period. Pollen germination percentage was ranged between 7.5% (MH26) and 61.25% (MH45). Pollen tube length was ranged between 7.09 mm (MH46) and 35.74 mm (MH55).
In the third experiment, two storage terms (six months and one year) were compared in mean pollen germination percentages and mean pollen tube lengths to determine statistical differences for each genotype by variance analysis (Table 3). Also proportional percentage changing in germination percentages and tube lengths from sixth month to one year was calculated for each genotype (Table 4 and Table 5).
Comparison of mean pollen germination percentages (%/˚C) and tube lengths (10 mm = 100.000 photo pixel/˚C) of 25 wild male genotypes in A. eriantha after one year storage
Germination percentage
Pollen tube length
Genotype
N1
Genotype
Genotype
N2
Result
MH10
4
0.00a
MH10
40
0.00a
MH30
4
0.00a
MH30
40
0.00a
MH31
4
0.00a
MH31
40
0.00a
MH34
4
0.00a
MH34
40
0.00a
MH43
4
0.00a
MH43
40
0.00a
MH57
4
0.00a
MH57
40
0.00a
MH58
4
0.00a
MH58
40
0.00a
MH66
4
0.00a
MH66
40
0.00a
MH26
4
7.50ab
MH46
40
7.09b
MH60
4
7.75ab
MH60
40
9.49b
MH46
4
10.25ab
MH26
40
11.83bc
MH48
4
12.25ab
MH48
40
16.78cd
MH67
4
18.25abc
MH56
40
18.46d
MH69
4
26.75abcd
MH69
40
18.53d
MH74
4
28.50bcde
MH74
40
19.07de
MH41
4
33.75bcde
MH45
40
19.21de
MH72
4
43.25cdef
MH41
40
21.44def
MH61
4
44.00cdef
MH70
40
23.21def
MH47
4
45.25cdef
MH47
40
25.48efg
MH55
4
46.75def
MH72
40
25.86fg
MH70
4
47.75def
MH22
40
26.41fg
MH56
4
49.75def
MH61
40
27.18fg
MH71
4
51.50def
MH71
40
29.98gh
MH22
4
55.50ef
MH67
40
35.23h
MH45
4
61.25f
MH55
40
35.74h
“N1” expresses total replication numbers; “N2” expresses total pollen grain numbers.
In the result, the differences between mean pollen germination percentages among six months and one year storages were found significant according to variance analysis for other genotypes except MH22, MH45, MH47, MH56, MH67, MH70, MH71, MH72 and MH74 genotypes. The proportional percentage changing of decreasing pollen germination percentage from sixth month to twelfth month in freeze storage for MH22, MH45, MH47, MH56, MH67, MH70, MH71, MH72 and MH74 were 2.63%, 9.93%, 12.14%, 5.24%, 30.48%, 31.54%, 25.63%, 23.79% and 24.50% respectively. In pollen tube length, only MH67 (from 35.46 mm to 35.23 mm) had not statistically significant difference with 0.9% changing from 6th month to 12th month.
Paired comparison of two storage terms in mean PG percentage (%) and PTL (10 mm) for each A. eriantha genotype
Genotype
Germination rate differences
SD
Tube length differences
SD
MH10
82.750*
3.637
43.542*
4.318
MH22
1.5
11.63
21.665*
3.588
MH26
43.500*
6.513
26.648*
2.774
MH30
45.750*
2.428
68.082*
3.76
MH31
55.000*
2.273
65.780*
3.804
MH34
56.750*
5.75
32.057*
1.63
MH41
40.750*
6.466
18.654*
2.369
MH43
79.500*
1.323
81.301*
3.453
MH45
6.75
12.592
16.025*
2.057
MH46
47.500*
7.38
39.273*
2.757
MH47
6.25
9.486
7.292*
2.513
MH48
12.750*
3.224
15.394*
2.411
MH55
19.000*
6.052
9.773*
3.183
MH56
2.75
10.423
21.538*
2.249
MH57
39.000*
8.377
26.530*
2.154
MH58
89.500*
1.041
88.958*
6.999
MH60
48.500*
4.56
38.947*
2.645
MH61
18.750*
5.483
17.159*
2.533
MH66
91.500*
1.708
86.529*
5.384
MH67
8
3.932
0.224
4.36
MH69
46.250*
5.202
34.505*
3.638
MH70
22
12.143
39.304*
3.129
MH71
17.75
7.526
21.117*
2.85
MH72
13.5
8.895
6.803*
2.365
MH74
9.25
3.934
7.145*
1.901
In the fourth experiment, differences between mean germination percentages and mean pollen tube lengths of variety were compared at the end of storage period (Table 6 and Table 7). Significant differences (p = 0.02) were found for both. Proportional percentage changing for PG and PTL were 60.52% and 69.58%, respectively. Average pollen germination percentage of variety decreased from 59.78% to 23.60% and pollen tube length of variety decreased from 48.78 mm to 14.84 mm from 6th month to 12th month.
Consequently, the germination percentages and tube lengths of genotypes decreased along long storage period. It was found in such investigation that the feijoa pollen partially lost its viability after been stored for 90 days in a freezer and had a great loss after 150 days [28] . In present work, Germination percentages and tube lengths of MH10, MH26, MH30, MH31, MH34, MH41, MH43, MH46, MH48, MH55, MH57, MH58, MH60, MH61, MH66 and MH69 genotypes significantly decreased from sixth month to twelfth month, some of them completely
Effect of storage time on pollen tube lengths (10 mm) of 25 wild A. eriantha genotypes
Genotype
PTL (10 mm)
Mean differences
(%) Differences
6 months
1 year
MH10
57.64
0
−57.64
100
MH22
48.08
26.41
−21.67
45
MH26
38.49
11.84
−26.65
69.24
MH30
68.08
0
−68.08
100
MH31
65.78
0
−65.78
100
MH34
32.06
0
−32.06
100
MH41
40.09
21.44
−18.65
46.60
MH43
81.30
0
−81.30
100
MH45
35.24
19.21
−16.03
45.49
MH46
46.37
7.09
−39.28
84.71
MH47
32.78
25.48
−7.30
22.27
MH48
32.18
16.78
−15.40
47.86
MH55
45.52
35.74
−9.78
21.68
MH56
40.00
18.47
−21.53
53.82
MH57
26.53
0
−26.53
100
MH58
88.96
0
−88.96
100
MH60
48.44
9.50
−38.94
80.39
MH61
44.35
27.19
−17.16
38.69
MH66
86.53
0
−86.53
100
MH67
35.56
35.24
−0.32
0.90
MH69
53.04
18.53
−34.51
65.06
MH70
62.52
23.22
−39.30
62.86
MH71
51.10
29.98
−21.12
41.33
MH72
32.67
25.86
−6.81
20.84
MH74
26.22
19.08
−7.14
27.23
Average
48.78
14.84
−33.94
69.58%
lost their viabilities. On the other hand, MH22, MH45, MH47, MH56, MH70, MH71, MH72 and MH74 genotypes had not significant differences in PG, although they had significant differences in PTL. Only MH67 had not significant differences in both pollen germination and pollen tube length, so these genotypes evaluated as vigor genotypes because the changing at their viability didn’t have statistically importance among freeze storage.
In this study, the variation in pollen traits of wild genotypes may be due to genetic diversity.
Effect of storage time on pollen germination percentages (%) of 25 wild A. eriantha genotypes
Genotype
PG (%)
Mean differences
(%) Differences
6 months
1 year
MH10
82.75
0
−82.75
100
MH22
57
55.5
−1.5
2.63
MH26
51
7.5
−43.5
85.29
MH30
45.75
0
−45.75
100
MH31
55
0
−55
100
MH34
56.75
0
−56.75
100
MH41
74.5
33.75
−40.75
54.70
MH43
79.5
0
−79.5
100
MH45
68
61.25
−6.75
9.93
MH46
57.75
10.25
−47.5
82.25
MH47
51.5
45.25
−6.25
12.14
MH48
25
12.25
−12.5
50.00
MH55
65.75
46.75
−19
28.90
MH56
52.5
49.75
−2.75
5.24
MH57
39
0
−39
100
MH58
89.5
0
−89.5
100
MH60
56.25
7.75
−48.5
86.22
MH61
62.75
44
−18.25
29.08
MH66
91.5
0
−91.5
100
MH67
26.25
18.25
−8
30.48
MH69
73
26.75
−46.25
63.36
MH70
69.75
47.75
−22
31.54
MH71
69.25
51.5
−17.75
25.63
MH72
56.75
43.25
−13.5
23.79
MH74
37.75
28.5
−9.25
24.50
Average
59.78
23.6
−36.18
60.52
Independent sample test (bootstrap), comparison of mean PTLs (10 mm = 100.000 photo pixel/˚C) of Actinidia eriantha variety along storage period
Pollen tube length
Mean differences
Bootstrap
deviation
Standard error
Sig.
95% Confidence
Low
Up
PTL
Variance equal
33.93880
−0.29668
4.64815
0.020
24.06459
45.67892
Variance not equal
33.93880
−0.29668
4.64815
0.020
24.06459
45.67892
Independent sample test (bootstrap), comparison of mean PG percentage (%/˚C) of Actinidia eriantha variety along storage period
Pollen germination percentage
Mean differences
Bootstrap
deviation
Standard error
Sig.
95% Confidence
Low
Up
PG
Variance equal
36.18000
−1.09757
6.31265
0.020
22.33194
49.53085
Variance not equal
36.18000
−1.09757
6.31265
0.020
22.33194
49.53085
4. Conclusion
This investigation demonstrates that germination ability of each wild genotype from Actinidia eriantha variety which was grown in the same origin area was different against long term freeze storage. Our investigation can be used as a basis source for further researches on genetic diversity of wild germplasm resources especially in A. eriantha variety as a plentiful source in natural distribution area.
Acknowledgements
This work was supported by the Special Science Foundation of Jiangxi Province (20143ACF60015, KJLD14026) & National Natural Scientific Fund (31360472).
Cite this paper
Umut Ahmet Seyrek,Xueyan Qu,Chunhui Huang,Junjie Tao,Min Zhong,Han Wu,Xiaobiao Xu, (2016) Influence of Storage Time on Pollen Traits in Actinidia eriantha. Agricultural Sciences,07,373-382. doi: 10.4236/as.2016.76039
NOTESReferencesNasib, A., Ali, K. and Khan, S. (2008) An Optimized and Improved Method for the in Vitro Propagation of Kiwifruit (Actinidia deliciosa) Using Coconut Water. Pakistan Journal of Botany, 40, 2355-2360.Qian, Y.Q. and Yu, D.P. (1991) Advances in Actinidia Research in China. Acta Horticulture, 297, 51-55.Liang C.F. ,et al. (1980)Outline of Taxonomy on Actinidia in China 1, 30-45.Khan, S.A. and Perveen, A. (2006) Germination Capacity of Stored Pollen of Solanum melongena L., (Solanaceae) and Their Maintenance. Pakistan Journal of Botany, 38, 917-920.Khan, S.A. and Perveen, A. (2006) Germination Capacity of Stored Pollen of Abelmoschus esculentus L. (Malvaceae) and Their Maintenance. Pakistan Journal of Botany, 38, 233-236.Bhat, Z.A., Dhillon, W.S., Shafi, R.H.S., Rather, J.A., Mir, A.H., Shafi, W., Rashid, R., Bhat, J.A., Rather, T.R. and Wani, T.A. (2012) Influence of Storage Temperature on Viability and in Vitro Germination Capacity of Pear (Pyrus spp.) Pollen. India Journal of Agricultural Science, 4, 128-137. http://dx.doi.org/10.5539/jas.v4n11p128Sharafi, Y. and Bahmani, A. (2011) Pollen Germination, Tube Growth and Longevity in Some Cultivars of Vitis vinifera L. African Journal of Microbiology Research, 5, 1102-1107.Kopp, R.F., Maynard, C.A., Niella, P.R.D., Smart, L.B. and Abrahamson, L.P. (2002) Collection and Storage of Pollen from Salix (Salicaceae). American Journal of Botany, 89, 248-252. http://dx.doi.org/10.3732/ajb.89.2.248Boughediri, L., Cerceau-Larrival, M.T. and Doré, J.C. (1995) Significance of Freeze-Drying in Long Term Storage of Date Palm Pollen. Grana, 34, 408-412. http://dx.doi.org/10.1080/00173139509429470Perveen A. ,et al. (2007)Pollen Germination Capacity, Viability and Maintanence of Pisium sativum L., (Papilionaceae) 2, 79-81.Lora, J., Oteyza, M.A.Pd., Fuentetaja, P. and Hormaza, J.I. (2006) Low Temperature Storage and in Vitro Germination of Cherimoya (Annona cherimola Mill.) Pollen. Pollen Scientia Horticulture, 108, 91-94.
http://dx.doi.org/10.1016/j.scienta.2005.12.003Weatherhead, M.A., Grout, B.W.W. and Henshaw, G.G. (2006) Advantages of Storage of Potato Pollen in Liquid Nitrogen. Biomed Life Science, 21, 331-334.Sukhvibul, N. and Considine, J.A. (1993) Medium and Long Term Storage of Anigozanthos manglesii (D. Don) Pollen. New Zealand Journal of Crop and Horticultural Science, 21, 343-347.
http://dx.doi.org/10.1080/01140671.1993.9513792Abreu, I. and Oliveira, M. (2004) Fruit Production in Kiwifruit (Actinidia deliciosa) Using Preserved Pollen. Australian Journal of Agricultural Research, 55, 565-569. http://dx.doi.org/10.1071/AR03211Borghezan, M., Clauman, A.D., Steinmacher, D.A., Guerra, M.P. and Orth, A.I. (2011) In Vitro Viability and Preservation of Pollen Grain of Kiwi (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev). Brazil Crop Breeding and Applied Biotechnology, 11, 338-344. http://dx.doi.org/10.1590/S1984-70332011000400007Vi?intin, L. and Bohanec, B. (2004) In Vitro Manipulation of Cucumber (Cucumis sativus L.) Pollen and Microspores: Isolation Procedures, Viability Tests, Germination, Maturation. Acta Biologica Cracoviensia Series Botanica, 46, 177-183.Nyomora, A.M.S., Brown, P.H., Pinney, K. and Polito, V.S. (2000) Foliar Application of Boron to Almond Trees Affects Pollen Quality. Journal of the American Society for Horticultural Science, 125, 265-270.Patel, R.G. and Mankad, A. (2012) In Vitro Pollen Germination—A Review. International Journal of Science and Research, 3, 305-307.Boavida, L.C. and McCormick, S. (2007) Temperature as a Determinant Factor for Increased and Reproducible in Vitro Pollen Germination in Arabidopsis thaliana. The Plant Journal, 52, 570-582.
http://dx.doi.org/10.1111/j.1365-313X.2007.03248.xImani, A., Kargar, M.H., Pireivatlou, S.P., Asgari, F. and Masomi, S.H. (2011) Evaluation of Germination Capacity of Stored Pollen of Almond and Peach. International Journal of Nuts and Related Sciences, 2, 68-72.Imani A. ,et al. (2012)Standardization of Different Media for in Vitro Pollen Germination of Almond and Evaluation of the Germination Capacity of Stored Pollen 11, 8843-8847.Soares, T.L., et al. (2008) In Vitro Germination and Viability of Pollen Grains of Banana Diploids. Crop Breeding and Applied Biotechnology, 8, 111-118.Qi, X.J., Zhang, S.L. and Fang, J.B. (2011) Effect of Culture Condition on Pollen Germination of Kiwifruit. Acta Agriculturae Zhejiangensis, 23, 528-532.Mondal, S. and Ghanta, R. (2012) Studies on in Vitro Pollen Germination of Helicteres isora Linn. Indian Journal of Plant Sciences, 1, 25-29.Youmbi, E., Fonkam, N.J.P., Tomekpe, K. and Fonbah, C. (2011) In Vitro Germination and Pollen Conservation of Some Musa Species. Asian Journal of Biotechnology, 3, 554-563. http://dx.doi.org/10.3923/ajbkr.2011.554.563Daher, F.B., Chebli, Y. and Geitmann, A. (2009) Optimization of Conditions for Germination of Cold-Stored Arabidopsis thaliana Pollen. Plant Cell Reports, 28, 347-357.Khan, S.A. and Perveen, A. (2008) Germination Capacity of Stored Pollen of Morus alba (Moraceae) and Their Maintenance. Pakistan Journal of Botany, 40, 1823-1826.Franzon, R.C., Corrêa, E.R. and do Carmo Bassols Raseira, M. (2005) In Vitro Pollen Germination of Feijoa (Acca sellowiana (Berg) Burret). Brazil Crop Breeding and Applied Biotechnology, 5, 229-233.
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