Orthogonal test design for optimizing culture medium for in vitro pollen germination of interspecific oil tea hybrids

Abstract

Oil Tea (Camellia oleifera) is an important woody edible oil plant in China. Oil Tea suffers from low rate of fruit set during production, which is related to poor pollination and fertilization. Pollen vigor is directly related to pollination and fertilization. Using the interspecific hybrid Y3 (C. grijsii × C. oleifera) as plant material, we studied the effects of sucrose, H₃BO₃, MgSO₄, and IAA on pollen germination using an orthogonal design to determine the best culture medium. Results indicated that pollen germination rates were significantly affected by medium components and ranged from 29.13% to 56.84%. Pollen tube length was the longest in the T5 medium surpassing the control group by 489.36 μm. MgSO₄ turned out to be the most important germination medium component having great effect on the pollen germination rate. The optimal culture medium to promote pollen tube growth of Oil Tea Y3 was: 1% agar, 150 g·L^-1 sucrose, 0.15 g·L^-1 H₃BO₃, 0.07 g·L^-1 MgSO₄, and 0.01 g·L^-1 IAA. The results of this paper may provide information for foliar application of Mg and IAA, which can improve pollen tube growth of Oil Tea in practice.

Key words
Oil Tea; pollen germination rate; pollen tube growth; Mg; IAA

INTRODUCTION

Camellia oleifera is a small evergreen tree species that belongs to the genus Camellia in the Theaceae family and produces edible oil from its seed (Xiong et al. 2019a). Tea oil is rich in unsaturated fatty acids and vitamin E and is known throughout the world as the “Oriental Olive Oil” (Gao et al. 2018). At present, the cultivated area of C. oleifera in China has exceeded 4.26 million hectares in 2016, but the production of tea oil is only 0.50 million tons (Qin et al. 2018). C. oleifera has self-incompatibility, so two or more are required for good pollination (Liao et al. 2014, Gao et al. 2015). Effective pollination mainly depends on good pollen vigor, which depends on the rate of pollen germination and the growth rate of pollen tubes (Ottaviano & Mulcahy 1989). Weak pollen vigor usually leads to lack of pollen germination or poor growth of pollen tubes, which are ineffective for fertilization (Xiong et al. 2016). Poor fertilization is responsible for fruit abortion, which severely impact yields of C. oleifera (Gao et al. 2017). Many reports have indicated that pollen vigor is one of the most important factors for the limitation of sexual reproduction of C. oleifera (Souza et al. 2017) resulting in low yields (Yuan et al. 2010). Therefore, an in vitro germination medium is helpful to study the reproduction process of C. oleifera in detail, which can also contribute to better understand and maybe improve the cultivation of Camellia.

Pollen performance has an important role in pollination, and pollen vigor and germination rate are vital factors for pollination and fertilization success (Melekber & Aysun 2014, Sunilkumar et al. 2013). Pollen vigor is influenced by many factors, including species, temperature and storage time, especially culture medium (Stanley & Linskens 1974, Franzon et al. 2005). Therefore, understanding the factors affecting pollen vigor could help with implementing management measures to improve fertilization and fruit yield (De-oliveira et al. 2016). In vitro pollen germination and tube growth are affected by nutrients and plant growth regulators (Maita et al. 2015, Muengkaew et al. 2016). Proper application of nutrients and regulators is an effective method to promote pollen germination and tube growth (Lin et al. 2017, Naik et al. 2016, Radovic et al. 2016), and is also beneficial for fruit production (Gao et al. 2012). Nevertheless, only a few studies have examined the effects of nutrients and regulators application on the promotion of pollen germination and on the increase of the rate of fruit set of C. oleifera (Gao et al. 2012, Tan et al. 2010, Yuan et al. 2010).

Therefore, in this study, a 10-treatment orthogonal experiment was designed to determine the optimal levels of various nutrients, and consequently develop the best culture medium for pollen germination and tube growth of hybrid clone Y3 (C. grijsii × C. oleifera).

MATERIALS AND METHODS

Plant materials

Camellia grijsii is one of the most important oil plants in the Theaceae family (Weng 1997), and is distributed in Hunan, Jiangsu, Fujian, Guizhou, Shanxi, Hubei, and Guangxi provinces (Weng 1997, Zou et al. 2013). It is a high-quality cultivated species of Camellia with a thick crown, thin peel, strong drought resistance, and high economic value (Lin & Hu 1981). C. grijsii flowers from February to April. Its fruit capsules are highly resistant to anthracnose disease (Colletotrichum camelliae Massee), which causes fruit capsules of C. oleifera to fall off prematurely (Xiong et al. 2019b). The fruit contains edible oil which is conducive to medicinal and nutritional use (Weng 1997).

Camellia oleifera ‘Huashuo’ is a new large-fruit and high-yield cultivar bred from a C. oleifera seedling in 2009, with an average fruit weight of 68.75 g, and a maximum fruit weight of 99.20 g. C. oleifera ‘Huashuo’ blooms from October to December. This new variety has the characteristics of a large fruit, high yield, and high photosynthetic efficiency (Tan et al. 2011).

In early March 2011, we hybridized pollen of C. oleifera ‘Huashuo’ (male parent) with C. grijsii (female parent) and harvested several hundred hybrid seeds in autumn. March 2012, we planted the hybrid seeds at the Camellia nursery of Central South University of Forestry and Technology. In 2016, the young trees started blooming. We screened for superior Y3 individuals from the F1 generation. We collected uncracked anthers of Y3 in mid-November, 2018 and stored them for two days in a refrigerator at 4 °C before testing.

Experimental design

We used an orthogonal design to optimize the culture medium for pollen germination of C. oleifera Y3. The number “4” in the design table indicates the four factors used, namely [A] sucrose, [B] H₃BO₃, [C] MgSO₄, and [D] IAA. The number “3” represents the three optimization levels or rates, and the number “9” represents nine pollen treatments from T1 to T9. The control group (CK) consisted of culturing pollen on a medium containing 1% agar. Tables I and II provide additional information of the test parameters.

Table I
Orthogonal experimental design, with 4 factors and 3 levels.

Level	Factor (g·L-1)
	(A) Sucrose (B) H3BO3 (C) MgSO4 (D) IAA
1	100	0.10	0.03	0.01
2	150	0.15	0.05	0.02
3	200	0.20	0.07	0.03

• Note: The numbers “1 ~ 3” indicate three levels. The factors affecting pollen viability are the concentrations of [A] Sucrose, [B] H₃BO₃, [C] MgSO₄ and [D] IAA.

Table II
Orthogonal test on pollen germination rate and pollen tube length of hybrid clone Y3 of Camellia oleifera.

Treatment	Factor (g·L-1)				Pollen germination rate (%)	Pollen tube length (μm)
	A Sucrose	B H3BO3	C MgSO4	D IAA
CK	0.00	0.00	0.00	0.00	12.10d	95.53d
T1	100	0.10	0.03	0.01	54.59ab	403.84bc
T2	100	0.15	0.05	0.02	41.00b	215.36c
T3	100	0.20	0.07	0.03	31.28bc	172.90cd
T4	150	0.10	0.05	0.03	42.39ab	383.90bc
T5	150	0.15	0.07	0.01	51.85ab	584.89a
T6	150	0.20	0.03	0.02	52.46ab	561.73ab
T7	200	0.15	0.03	0.02	56.84a	560.76ab
T8	200	0.20	0.05	0.03	29.13bc	218.04c
T9	200	0.10	0.07	0.01	32.37bc	420.50b

• Note: Pollen germination rate and pollen tube length were measured after 2 hours of culture. Within columns, different letters indicate significant differences at P0.05.

Pollen culture

We weighed the reagents and labelled them in sequence and dissolved them by adding quantified distilled water. In order to sufficiently dissolve the agar and its content, we heated the mixture in a microwave oven, then cooled it at room temperature. After cooling the medium, we dropped the medium onto a microscope slide using a dropper. When the medium became semi-solid, we used brushes to scatter pollen on the surface of the medium (Xiong et al. 2016). We placed the microscope slides in a culture dish with wet filter paper and incubated them in a constant temperature incubator at 25 ^oC for 2 h in the dark. Each treatment consisted of 3 replicates.

Measurements

Pollen germination was monitored using a BX-53 microscope (Olympus, Tokyo, Japan). For each sample (slide), we randomly selected 5 optical fields containing no less than 50 pollen grains each (Tan et al. 2010, Yuan et al. 2010). We then counted the number of germinated pollen grains to determine the germination rate. The standard for pollen germination is that the length of pollen tube must exceed its diameter (Bryhan & Serdar 2008). Pollen germination rate (%) is equal to the number of germinated pollen grains divided by the total number of pollen grains (Tan et al. 2010). Pollen tube length was measured using the image processing software Image J (National Institutes of Health, Bethesda, USA). For each treatment, the length of 60 germinated pollen tubes were measured and used to calculate the average length of pollen tube.

Statistical analysis

We used SPSS 19.0 software (IBM company, New York, USA) to analyze the data, and to test the effects of four media on pollen germination rate and pollen tube length using a one-way analysis of variance (ANOVA). Significant differences among means were assessed using Duncan’s multiple comparison at p ≤ 0.05. Figures were drawn using Origin Pro8.5 software (Origin Lab company, Northampton, USA).

RESULTS

Pollen germination rate

Compared to the control media (CK), pollen germination rate was significantly higher in all tested treatment combinations. Germination rate was highest in T7 at 56.84%, which was close to that of T1 (54.59%), T5 (51.85%) and T6 (52.46%). Compared with the germination rate of other three factors, there was relatively low statistical difference between the germination rate of T1 (low sugar concentration) and T4, T5, T6 (medium sugar concentration) or T7 (high sugar concentration), which indicates that the effect of sucrose on pollen germination rate is a little similar under these three concentrations (Table IV). The decreasing order of the effect of each treatment on promoting pollen germination of hybrid clone Y3 was: T7 (56.84%) T1 (54.59%) T6 (52.46%) T5 (51.85%) T4 (42.39%) T2 (41.00%) T9 (32.37%) T3 (31.28%) T8 (29.13%) CK (12.10%) (Table II, Figure 3). When compared to the relative order of the R-values (R_C R_D R_B R_A, Table III), we found that the influence of the 4 factors on hybrid clone Y3 of C. oleifera pollen germination rates occurred in the following order: MgSO₄ IAA H₃BO₃ sucrose concentration. The ideal culture concentration for pollen germination rate was sucrose at 200g·L^-1, H₃BO₃ at 0.15g·L^-1, MgSO₄ at 0.03g·L^-1and IAA at 0.02g·L^-1, respectively. Interestingly, in terms of its effect on germination, medium T1 was almost as good as medium T7 (only 2% lower pollen germination), even though its recipe contains half the amount of sucrose and IAA. Thus, T1 is more economically friendly.

Figure 3
Pollen germination and pollen tube length after incubation for 2 h in Camellia oleifera hybrid clone Y3. For symbol T1~CK, represents different treatments of pollen, refer to Table II.

Table III
Range analysis for pollen germination rate and pollen tube length in Camellia oleifera hybrid clone Y3.

		(A) sucrose	(B) H3BO3	(C) MgSO4	(D) IAA
Pollen germination rate (%)	K1	126.87	129.35	163.89	138.81
	K2	146.70	149.69	112.52	150.30
	K3	118.34	112.87	115.50	102.80
	X1	42.29	43.12	54.63	46.27
	X2	48.90	49.90	37.51	50.10
	X3	39.45	37.62	38.50	34.27
	R	9.45	12.28	17.12	15.83
Pollen tube length (μm)	K1	792.10	1208.24	1526.33	1409.23
	K2	1530.52	1361.01	817.30	1337.85
	K3	1199.30	952.67	1178.29	774.84
	X1	264.03	402.75	508.78	469.74
	X2	510.17	453.67	272.43	445.95
	X3	399.77	317.56	392.76	258.28
	R	246.14	136.11	236.35	211.46

• Note: Ki was obtained by summing the total number of columns corresponding to level i, that is, the sum of pollen germination rate or pollen tube length corresponding to three concentration levels of each factor. Xi is the mean value of Ki (i.e., Ki/3), and R is the maximum Xi minus the minimum Xi.

Table IV
Variance analysis for the pollen germination rate and pollen tube length in Camellia oleifera hybrid clone Y3.

	Factor	SS	DF	F	p	Significance
Pollen germination rate(%)	sucrose	3094.022	3	7.941	0.001	*
	H3BO3	3350.616	3	9.307	0.001	*
	MgSO4	4334.112	3	17.581	0.001	*
	IAA	3910.728	3	13.389	0.001	*
Pollen tube length (μm)	sucrose	1823941.580	2	18.414	0.001	*
	H3BO3	567523.617	2	5.011	0.008	*
	MgSO4	1675928.678	2	16.639	0.001	*
	IAA	1610034.936	2	15.868	0.001	*

• Note: DF = Degree freedom. F value = Mean square of between groups / within groups. The p-value indicates a significant difference. SS = Sums of squares between groups. * Significant at the 0.01 level.

Pollen tube length

According to Table II, pollen tube length was longest in T5 (584.89 μm), and shortest in T3 (172.90 μm). The order of effect of each treatment on promoting pollen tube growth was T5 (584.89 μm) T6 (561.73 μm) T7 (560.76 μm) T9 (420.50 μm) T1 (403.84 μm) T4 (383.90 μm) T8 (218.04 μm) T2 (215.36 μm) T3 (172.90 μm) CK (95. 53μm) (Table II, Figure 3). Among these treatments, T5 was the best medium for promoting pollen tube growth of hybrid clone Y3 in this experiment. The nutrient elements and proportion for T5 are sucrose 150g·L^-1, H₃BO₃ 0.15g·L^-1 , MgSO₄ 0.07g·L^-1, and IAA 0.01g·L^-1 . Sucrose, H₃BO₃, MgSO₄ and IAA were the main factors affecting the hybrid clone Y3 pollen tube growth (Table III ), with R-values in the order of RA RC RD RB (Table III ). The influence of the 4 factors on the pollen tube length was: sucrose MgSO₄ IAA H₃BO₃ concentration.

Figure 1
Influence of 4 factors on pollen germination rate in Camellia oleifera hybrid clone Y3. A: sucrose concentration; B: H₃BO₃ concentration; C: MgSO₄ concentration; D: IAA concentration. Data are based on values listed in Table II.

DISCUSSION

Pollen germination is influenced by several factors including nutrition (Imani & Talaie 2006, Bal & Ubak 2005, Maita & Sotomayor 2015, Muengkaew et al. 2016) and plant growth regulators (Kovaleva et al. 2005, Wu et al. 2008). Nutrients, such as sucrose, boron, and magnesium have obvious effects on pollen growth (Chen et al. 2009, Liu et al. 2013, Naik et al. 2016). Sucrose plays an extremely important role and is a significant nutrient for pollen germination (Fei & Nelson 2003, Liu et al. 2013). It helps to protect membranes and preserve protein structure from desiccation, because pollen grains lose water rapidly after release (Kosel et al. 2018). Huang et al. (2011) reported that pollen germination of C. oleifera was best when sucrose concentration was 150 g·L^-1 and began to decrease at 200 g·L^-1. The optimum sucrose concentration range for promoting pollen germination is between 100 g·L^-1 and 150 g·L^-1 . Similar results were observed in our study, adding 100-150 g·L^-1 sucrose significantly promoted pollen germination, and was the best at a concentration of 150g·L^-1.

Boron plays an important role in stimulating the growth of pollen grains and pollen tubes (Izzet et al. 2010, Lee et al. 2009, Nyomora et al. 2000). Yuan et al. (2010) found that pollen germination of C. oleifera was significantly promoted at boric acid concentrations of 0.02-0.10 g·L^-1 and was best at 0.10 g·L^-1. Tanmoy et al. (2018) reported that boric acid concentration of 0.8 g·L^-1 was a suitable medium for guava pollen germination and pollen tube growth. Lee et al. (2009) stated that the germination rate and tube growth of pear were highest at a boron concentration of 0.2 g·L^-1. Our results were similar to those of a study by Jiang et al. (2010), who found that boric acid promoted pollen germination availably within the range of 0.10 g·L^-1 to 0.15 g·L^-1.

Figure 2
Influence of 4 factors on pollen tube length in Camellia oleifera hybrid clone Y3. A: sucrose concentration; B: H₃BO₃ concentration; C: MgSO₄ concentration; D: IAA concentration. Data are based on values listed in Table II.

Magnesium has been found to play an important role in pollen germination (Chen et al. 2009). Magnesium transport is significantly related to pollen homeostasis and germination (Chen et al. 2009). Yuan et al. (2010) found that as the concentration of magnesium increased from 0.02 to 0.05 g·L^-1, pollen germination was promoted in C. oleifera. Han et al. (2014) reported that magnesium inhibited pollen germination and pollen tube growth in Ziziphus jujuba to some extent, and the degree of inhibition decreased first and then increased with the increase in concentration. When the magnesium concentration was in the range of 0.01 to 0.05 g·L^-1, it promoted both pollen germination and pollen tube growth. According to our results, magnesium played the important role in pollen development of C. oleifera Y3 and the best concentration for pollen grain germination was 0.03 g·L^-1. Similar results were obtained by Yuan et al. (2010) and Han et al. (2014). However, Jiang et al. (2010) observed that magnesium had no significant effect on pollen germination in Clivia miniata. The reason for this difference may be due to variety and genotype differences of plant material.

In general, with the increase in the concentration of growth regulators, the germination rate of pollen in vitro will fluctuate (Gokbayraka & Engin 2016). IAA, a plant growth regulator, could promote pollen germination at low concentrations, but can inhibit growth at higher concentrations (Kovaleva et al. 2005). It has also been suggested that IAA at appropriate concentrations promotes pollen tube growth (Abdelgadir et al. 2012, Tian et al. 1996). Tian et al. (1996) reported that 0.005 g·L^-1 IAA significantly promoted pollen tube growth, while 0.10 g·L^-1 IAA completely inhibited pollen germination. Tan et al. (2010) observed that pollen germination rate was highest when the concentration of IAA was 0.005 g·L^-1. From their research, IAA promoted pollen germination in the range of 0.01 to 0.02 g·L^-1. In our study, 0.01 to 0.02 g·L^-1 of IAA increased the germination rate. This result was agreement with that of Tan et al. (2010). Thus, our results may provide a reference for pollen germination and pollen tube growth in C. oleifera.

CONCLUSIONS

Pollen grain performance, including pollen grain germination and tube growth rate, is an important component of successful pollination and fertilization for C. oleifera. In this paper, pollen tube length was affected by added components in the following decreasing order of effect: sucrose MgSO₄ IAA H₃BO₃. The optimum medium for pollen tube growth was 1% agar, 150 g·L^-1 sucrose, 0.15g·L^-1 H₃BO₃, 0.07 g·L^-1 MgSO₄, and 0.01 g·L^-1 IAA. These results may provide useful information for promoting pollen tube growth of C. oleifera in practice.

ACKNOWLEDGMENTS

This work has been supported by the Major Scientific and Technology Special Program in Hunan Province (No. 2018NK1030), the National Key Research and Development Program Project of China (No. 2018YFD1000603) and the National Forestry Public Welfare Foundation of China (No. 201504705).

REFERENCES

• Abdelgadir HA, Johnson SD VanStaden J. 2012. Pollen viability, pollen germination and pollen tube growth in the biofuel seed crop Jatropha curcas (Euphorbiaceae). S Afr J Bot 79: 132-139.
• Bal U Abak K. 2005. Effects of sucrose, maltose, pH and phloroglucinol on the germination of globe artichoke pollen in vitro. Eur J Hortic Sc 70(3): 142-148.
• Bryhan N Serdar U. 2008. Assessment of pollen viability and germinability in some European chestnut genotypes (Castanea sativa L.). Hort Sci (Prague) 35(4): 171-178.
• Chen J, Li LG, Liu ZH, Yuan YJ, Guo LL, Mao DD, Tian LF, Chen LB, Luan S Li DP. 2009. Magnesium transporter AtMGT9 is essential for pollen development in Arabidopsis. Cell Research 19(7): 887-898.
• DE-OLIVEIRA LF, RUIZ C, DE-OLIVEIRA AF, PINO R DIAS E. 2016. Establishment of growth medium and quantification of pollen grains of olive cultivars in Brazil’s subtropical areas. Bragantia 75(1): 26-32.
• Fei S Nelson E. 2003. Estimation of pollen viability, shedding pattern, and longevity of creeping bentgrass on Artificial media. Crop Sci 43(6): 2177-2181.
• Franzon RC, Correa ER Raseira MCB. 2005. In vitro pollen germination of feijoa (Acca sellowiana (Berg) Burret). Crop Breed Appl Biot 5: 229-233.
• Gao C, Yang R Yuan DY. 2018. Structural characteristics of the mature embryo sac of Camellia oleifera. Nord J Bot 36: e01673.
• GAO C, Yang R YUAN DY. 2017. Characteristics of Developmental Differences between Fertile and Aborted Ovules in Camellia oleifera. J Am Soc Hortic Sci 142(5): 330-336.
• Gao C, Yuan DY, Yang Y, Wang B, Liu D Zou F. 2015. Pollen Tube Growth and Double Fertilization in Camellia oleifera. J Am Soc Hortic Sci 140(1): 12-18.
• GAO C, YUAN DY, YUAN J, LIAO T, ZOU F DUAN WH. 2012. The effect of spraying nutrient elements and growth regulators at bloom on fruit setting rate of Camellia oleifera. Acta Agriculture Universitatis Jiangxiensis 34(3): 505-510. (in Chinese with English abstract).
• Gokbayraka Z Engin H. 2016. Effect of plant growth regulators on in vitro pollen germination of grapevine cultivars. ISHS Acta Hortic: III Balkan Symposium on Fruit Growing 1139: 405-408.
• Huang YF, Wu XH, He ME, Ding J An X. 2011. Studies on the pollen storage and viability of 3 oil-tea species. J Fujian Col Forest 31(1): 56-59. (in Chinese with English abstract).
• Han ZQ, Yuan DY, Chen WT, Li CX Wei J. 2014. Effects of different nutrient elements on pollen germination and tube growth in Ziziphus jujube Mill. Acta Agriculturae Universitatis Jiangxiensis 36(2): 357-363. (in Chinese with English abstract).
• Imani A Talaie AR. 2006. A study on the relationship between pollen germinability and some of its elements in almond. Acta Horticult 726: 399-402.
• Izzet A, Bekir EAk Kamil S. 2010. Effects of boron and gibberellic acid on in vitro pollen germination of pistachio (Pistacia vera L.). Afri J Biotechnol 32(9): 5126-5130.
• Jiang C, Wang C Lei JJ. 2010. Determination of pollen viability and screening of storage methods in Clivia miniata Regel. Acta Agriculturae Boreali-Occidentalis Sinica 19(5): 157-161. (in Chinese with English abstract).
• Kovaleva LV, Zakharova EV, Minkina YV, Timofeeva GV Andreev IM. 2005. Germination and in vitro growth of petunia male gametophyte are affected by exogenous hormones and involve the changes in the endogenous hormone level. Russ J Plant Physiol 52(4): 521-526.
• Kosel J, Vižintin L, Majer A Bohanec B. 2018. Staining for viability testing, germination and maturation of Sambucus nigra L. pollen in vitro. Biotechnic Histochemistry Official Publication of the Biological Stain Commission 93(4): 1-9.
• Lee SH, Kim WS Han TH. 2009. Effects of post-harvest foliar boron and calcium applications on subsequent season’s pollen germination and pollen tube growth of pear (Pyrus pyrifolia). Sci Horticul 122(1): 77-82.
• Liao T, Yuan DY, Zou F, Gao C, Yang DY, Zhang L Tan XF. 2014. Self-Sterility in Camellia oleifera may be due to the Prezygotic Late-Acting Self-Incompatibility. PLoS ONE 9(6): e99639.
• Lin YY, Wang Y, Amjad I, Shi P, Li J, Yang YD Lei XT. 2017. Optimization of culture medium and temperature for the in vitro germination of oil palm pollen. Sci Horticult 220: 134-138.
• Lin SL Hu SY. 1981. Preliminary observation on flower bud differentiation of Camellia grijsii Hance. Journal of Hunan Agricultural University (Natural Sciences) (2): 45-48. (in Chinese with English abstract).
• Liu LY, Huang LY Li Y. 2013. Influence of boric acid and sucrose on the germination and growth of Areca pollen. Am J Plant Sci 4: 1669-1674.
• Maita S Sotomayor C. 2015. The effect of three plant bioregulators on pollen germination, pollen tube growth and fruit set in almond [Prunus dulcis (Mill.)D.A. Webb] cvs. Non Pareil and Carmel. Electron J Biotechnol 18: 381-386.
• Muengkaew R, Chaiprasart P Wongsawad P. 2016. Calcium-boron addition promotes pollen germination and fruit set of mango. Int J Fruit Sci 17(2): 147-158.
• Melekber S Aysun C. 2014. In vitro pollen viability and pollen germination in cherry laurel (Prunus laurocerasus L.). The Scientific World Journal http://dx.doi.org/10.1155/2014/657123
  » http://dx.doi.org/10.1155/2014/657123
• Nyomora AMS, Brown PH, Pinney K Polito VS. 2000. Foliar application of boron to almond trees affects pollen quality. J A Soc Hortic Sci 125(2): 265-270.
• Naik A, Akhtar S, Chattopadhyay A, Thapa U Hazra P. 2016. In vitro teasle gourd pollen germination and pollen tube development as affected by sucrose, boric acid, and inorganic salts. Int J Veg Sci 22(2): 209-216.
• Ottaviano E Mulcahy DL. 1989. Genetics of angiosperm pollen. Adv Genet 26: 1-64.
• Qin SY, Rong J, Zhang WJ Chen JK. 2018. Cultivation history of Camellia oleifera and genetic resources in the Yangtze River Basin. Biodiversity Science 26(4): 384-395. (in Chinese with English abstract).
• Radovic A, Nikolic D, Milatovic D, Zivkovic B Stevanovic N. 2016. The effect of plant hormones on pollen germination and pollen tube growth of almond cultivars. ISHS Acta Horticulturae 1139: III Balkan Symposium on Fruit Growing, p. 375-379.
• Sunilkumar K, Mathur RK, Sparjanbabu DS Reddy AGK. 2013. Pollen viability and vigour in interspecific hybrids (E. guineensis × E. oleifera) of oil palm. Journal of Plantation Crops 41(1): 91-94.
• Stanley RG Linskens HF. 1974. Pollen biochemistry management. Berlin: Heidelberg.
• SOUZA FBMD, PIO R, MARAISA H, ZAMBON CR REIGHARD GL. 2017. Boric acid in germination of pollen grains and fruit set of peach cultivars in subtropical region. Rev Cienc Agron 48(483): 496-500.
• Tanmoy S, Sushanta KS Sathish V. 2018. Effect of Sucrose and Boric Acid on in-vitro Pollen Germination of Guava (Psidium guajava) Varieties. Advances in Research 15(1): 1-9.
• Tan XF, Yuan DY, Yuan J Liao T. 2010. Pollen germination in Camellia oleifera with ascorbic acid and plant growth regulators. Journal of Zhejiang Forestry College 27(6): 941-944. (in Chinese with English abstract).
• Tan XF, Yuan DY, Yuan J, Zou F, Xie P, Su Y, Yang DT Peng JT. 2011. An Elite Variety: Camellia oleifera ‘Huashuo’. Scientia Silvae Sinicae 47(12): 184-185. (in Chinese with English abstract).
• Tian XS, Feng L Pan RC. 1996. Effects of IAA on Pollen Tube Elongation and Endogenous cAMP Content in Cucumber. Journal of South China Normal University (Natural Science Edition) (2): 90-92. (in Chinese with English abstract).
• Weng Y. 1997. Studies of Camellia grijsii Hance. Commonwealth Forestry Review 76(2): 132-133.
• Wu JZ, Lin Y, Zhang XL, Pang DW Zhao J. 2008. IAA stimulates pollen tube growth and mediates the modification of its wall composition and structure in Torenia fournieri. J Exp Bot 59(9): 2529-2543.
• Xiong H, Zou F, Yuan DY, Zhang X Tan XF. 2016. Orthogonal test design for optimizing the culture medium for in vitro pollen germination of feijoa (Acca sellowiana cv. Unique). N Z J Crop Hortic Sci 44(3): 192-202.
• Xiong H, Zou F, Yuan DY, Tan XF, Yuan J, Liao T Niu GH. 2019a. Comparison of self- and cross-pollination in pollen tube growth, early ovule development and fruit set of Camellia grijsii. Int J Agric Biol 21(4): 819-826.
• Xiong H, Chen P, Zhu ZJ, cHEN y, zOU F YUAN DY. 2019b. Morphological and cytolpgical characterization of petaloid-type cytoplasmic male sterility in Camellia oleifera. Hort Sci 54(7): 1149-1155.
• Yuan DY, Wang R, Yuan J, Liao T, Cui X Cai L. 2010. Effects of different nutrient elements and ratios on pollen germination rate of Camellia oleifera. Journal of Fujian Agricultural and Forestry University (Natural Science Edition) 39(5): 471-474. (in Chinese with English abstract).
• ZOU F, YUAN DY, DUAN J, TAN XF ZHANG L. 2013. A study of microsporgenesis and male gametogenesis in Camellia grijsii Hance. Adv J Food Sci Technol 5: 1590-1595.

Publication Dates

• Publication in this collection
  26 Feb 2021
• Date of issue
  2021

History

• Received
  11 Apr 2019
• Accepted
  20 Nov 2019