1. Introduction

Rice ( Oryza sativa L.) is one of the most important cereal crops and serves as a staple food for more than half of the world’s population. Increasing global population, climate variability, emerging pathogens, and declining agricultural resources have intensified the need for the development of improved rice cultivars with enhanced productivity, stress tolerance, and nutritional quality [1]. In recent years, plant genetic engineering has emerged as an effective strategy for introducing desirable agronomic traits into rice plants, including resistance to insects, diseases, herbicides, drought, and salinity stress [2]. Transgenic technology has significantly contributed to crop improvement programs by enabling the transfer of specific genes across species barriers. However, along with agronomic performance, evaluation of reproductive fitness is essential for assessing the stability and biosafety of transgenic plants [3]. Reproductive fitness determines the ability of plants to produce viable gametes, achieve successful fertilization, and maintain stable inheritance of introduced genes across generations. Any unintended effects resulting from transgene integration may alter pollen development, fertility, flowering behavior, or seed production [4].

Pollen viability is considered one of the most reliable indicators of male reproductive success in flowering plants. Viable pollen grains possess functional cytoplasmic contents and are capable of successful germination and fertilization, whereas non-viable pollen grains fail to germinate or produce defective pollen tubes [5]. Assessment of pollen viability therefore plays a crucial role in plant breeding, hybrid seed production, biosafety evaluation, and characterization of genetically modified crops [6]. Several staining methods have been developed for pollen viability analysis, among which Alexander staining remains one of the most widely accepted cytological techniques due to its simplicity, accuracy, and ability to differentiate viable and aborted pollen grains effectively [7]. In this staining method, viable pollen grains typically appear dark red or purple because of intact protoplasmic contents, while non-viable pollen grains exhibit greenish or faint coloration due to the absence of cellular integrity [8]. Microscopic examination of stained pollen grains provides rapid and reliable estimation of fertility status in transgenic plants, pollen germination tests provide functional evidence regarding the fertilization potential of pollen grains. Successful pollen germination and pollen tube elongation are essential prerequisites for fertilization and seed formation [9]. Reduced germination ability may indicate physiological abnormalities, impaired metabolic activity, or adverse effects associated with transgene insertion and tissue culture-mediated transformation procedures [10]. Male sterility is another important reproductive characteristic frequently evaluated in transgenic plants. Male sterile lines exhibit reduced or absent functional pollen production and are particularly important in hybrid breeding programs [11]. Cytological and morphological examination of pollen grains can help identify sterility-associated abnormalities and reproductive impairments in genetically modified lines [12]. Previous studies have demonstrated that certain transgenic crops may exhibit altered reproductive traits due to insertional mutagenesis, somaclonal variation, epigenetic modifications, or changes in gene expression patterns [13]. Therefore, detailed evaluation of pollen fertility and viability is essential for confirming reproductive stability and environmental safety before commercialization of transgenic rice cultivars [14]. The present study aimed to analyze the reproductive fitness of T1 rice transgenic lines through pollen viability, fertility, male sterility, and pollen germination assays. Comparative evaluation between transgenic and untransformed control plants was conducted using Alexander staining and microscopic analysis. The study provides important insights into the reproductive behavior and stability of transgenic rice lines and contributes to future breeding and biosafety assessment programs.

2. Materials and Methods

2.1 Plant Materials and Growth Conditions

T1 generation rice transgenic lines along with untransformed control plants were used in the present investigation. The plants were maintained under controlled greenhouse conditions with proper irrigation, nutrient management, and photoperiodic regulation to ensure uniform growth and reproductive development. Healthy plants showing normal vegetative growth were selected for pollen viability and fertility analysis during the flowering stage.

2.2 Collection of Floral Samples

Freshly opened flowers at anthesis were collected from both transgenic and control rice plants during morning hours when pollen viability is generally highest. Spikelets were carefully detached using sterile forceps, and mature anthers were isolated for cytological examination. The collected floral samples were immediately processed to avoid desiccation and loss of pollen viability.

2.3 Pollen Viability Assay Using Alexander Staining

Pollen viability analysis was carried out following the Alexander staining protocol with slight modifications. Fresh pollen grains were gently released from mature anthers onto clean microscopic glass slides using dissecting needles. A drop of Alexander stain was added to the pollen samples, followed by careful placement of a coverslip. The prepared slides were incubated briefly to allow proper staining and then examined under an Olympus light microscope at different magnifications. Viable pollen grains appeared dark red or purple due to the presence of intact cytoplasmic contents, whereas non-viable or aborted pollen grains exhibited faint green or bluish coloration with shriveled morphology. For each sample, multiple microscopic fields were examined, and at least 300 pollen grains were counted randomly to determine pollen viability percentage. Photomicrographs were captured for comparative analysis between transgenic and control plants.

The percentage of pollen viability was calculated using the following equation:

2.4 Evaluation of Pollen Fertility and Male Sterility

Pollen fertility was evaluated based on staining response, pollen morphology, and frequency of viable pollen grains. Fertile pollen grains exhibited uniform size, spherical morphology, and intense staining characteristics, whereas sterile pollen grains appeared collapsed, irregularly shaped, and weakly stained. Male sterile plants were identified through microscopic examination of pollen grains and floral structures. Plants exhibiting significantly reduced viable pollen production and poor pollen development were categorized as partially or completely male sterile. Comparative observations were recorded between transgenic and control plants to determine possible effects of genetic transformation on male reproductive characteristics.

2.5 In Vitro Pollen Germination Assay

Pollen germination tests were conducted to evaluate the functional capability of pollen grains. Fresh pollen grains were inoculated onto germination medium containing suitable concentrations of sucrose, boric acid, calcium nitrate, and agar. The inoculated plates were incubated under controlled laboratory conditions for a specific duration to facilitate pollen tube emergence. After incubation, the germinated pollen grains were observed under an Olympus microscope. Pollen grains with pollen tube length greater than the pollen diameter were considered germinated. Multiple microscopic fields were examined to determine germination percentage.

The pollen germination percentage was calculated using the formula:

2.6 Microscopic Observation and Imaging

All stained pollen grains and germination assays were observed using an Olympus compound microscope. Representative images of viable, non-viable, germinated, and sterile pollen grains were photographed for documentation and comparative analysis.

2.7 Statistical Analysis

All experiments were performed in triplicates, and the obtained data were expressed as mean ± standard deviation (SD). Statistical comparisons between transgenic and control plants were analyzed using standard statistical procedures. Graphical representation of pollen viability, fertility, and germination data was prepared using suitable software tools.

3. Results and Discussion

3.1 Morphological Observation of Pollen Grains

Microscopic examination revealed distinct differences between viable and non-viable pollen grains in transgenic and control rice plants. Viable pollen grains were spherical, uniformly stained, and structurally intact, while non-viable pollen grains appeared shrunken, irregular, and weakly stained. The Alexander staining protocol effectively differentiated fertile and aborted pollen grains, allowing rapid assessment of reproductive performance. The untransformed control plants exhibited a comparatively higher proportion of viable pollen grains with normal morphology. In contrast, some transgenic lines displayed slight reductions in pollen viability and increased frequency of malformed pollen grains, suggesting that genetic transformation may influence pollen development and reproductive stability.

Figure 1: Pollen grains from freshly opened flowers collected from T1 rice transgenic and untransformed control plants stained using Alexander staining protocol and observed under Olympus microscope.

3.2 Evaluation of Male Sterility

Male sterility analysis demonstrated significant variation among the transgenic lines. Certain transgenic plants exhibited reduced pollen production, abnormal pollen morphology, and poor staining intensity compared with the control plants. Sterile pollen grains appeared collapsed and lacked normal cytoplasmic contents. The occurrence of male sterility in transgenic plants may result from insertional effects of transgenes, somaclonal variation generated during tissue culture, or altered expression of genes associated with pollen and anther development. Reduced pollen fertility may adversely affect reproductive success, seed set, and stable inheritance of introduced genes. Despite the occurrence of partial sterility in some lines, several transgenic plants maintained acceptable fertility levels comparable to the control plants, indicating successful reproductive adaptation following transformation.

3.3 Evaluation of Pollen Fertility and Viability

Comparative assessment of pollen fertility revealed differences between transgenic and control rice plants. The control plants exhibited high pollen viability and uniform staining patterns, whereas transgenic lines showed varying fertility responses. Some transgenic plants maintained high pollen viability, while others exhibited moderate reductions in viable pollen frequency. The reduced fertility observed in certain transgenic lines may be associated with physiological stress, genomic rearrangements, or altered metabolic activity caused by transgene insertion. Similar observations have been reported in several genetically modified crop species where reproductive alterations were linked to tissue culture-mediated transformation processes.

Pollen fertility is a critical determinant of reproductive success and stable transmission of genetic material across generations. Therefore, maintenance of viable and functional pollen grains in transgenic plants is essential for breeding programs and biosafety evaluations.

3.4 In Vitro Pollen Germination Assay

The pollen germination assay demonstrated the functional capability of pollen grains to initiate pollen tube development under in vitro conditions. Germinated pollen grains developed elongated pollen tubes, whereas non-germinated pollen grains failed to show tube emergence. The control plants exhibited relatively higher pollen germination percentages, indicating strong reproductive competence. Some transgenic lines also showed satisfactory germination ability, suggesting that the introduced genetic modifications did not significantly impair pollen functionality. However, male sterile plants and partially sterile lines displayed markedly reduced germination frequencies. Reduced pollen germination may result from impaired membrane integrity, altered enzymatic activity, or metabolic disturbances affecting pollen tube growth. Since pollen germination is directly related to fertilization efficiency and seed development, lower germination rates may negatively influence reproductive fitness in certain transgenic lines.

3.5 Reproductive Fitness of T1 Transgenic Rice Lines

Overall, the present investigation demonstrated that pollen viability assays, fertility analysis, and pollen germination tests are effective tools for evaluating reproductive fitness in transgenic rice plants. The majority of transgenic lines retained moderate to high reproductive performance, while a few lines exhibited partial sterility and reduced pollen functionality. The observed variability among transgenic plants may be attributed to transgene integration sites, copy number variation, physiological stress, and tissue culture-induced genetic instability. The findings emphasize the importance of detailed reproductive assessment during the development and characterization of genetically modified crops. These results also support the use of pollen-based cytological techniques for biosafety evaluation, breeding selection, and identification of stable transgenic lines suitable for future agricultural applications.

4. Conclusion

The present study successfully evaluated the reproductive fitness of T1 rice transgenic lines through comprehensive pollen viability, fertility, male sterility, and pollen germination assays. Alexander staining proved to be an effective and reliable method for differentiating viable and non-viable pollen grains and enabled detailed cytological assessment of reproductive performance in transgenic rice plants. Microscopic observations demonstrated distinct variations in pollen morphology, staining intensity, and germination behavior between transgenic and untransformed control plants. The results revealed that several transgenic rice lines maintained satisfactory pollen viability and germination capacity comparable to the control plants, indicating stable reproductive performance and functional male fertility. However, certain transgenic lines exhibited reduced pollen viability, abnormal pollen morphology, and partial male sterility, suggesting that genetic transformation and tissue culture procedures may influence reproductive development. Variability in reproductive traits among the transgenic lines may be associated with transgene insertional effects, physiological stress, genomic rearrangements, or altered gene expression patterns.

The pollen germination assay further confirmed the functional competence of viable pollen grains and highlighted the importance of pollen tube development in successful fertilization and seed formation. Reduced germination observed in some lines indicates possible impairment in pollen functionality and reproductive efficiency, the findings demonstrate that pollen-based assays provide valuable information regarding reproductive stability and biosafety evaluation of transgenic rice plants. The study contributes important baseline data for the characterization of genetically modified rice lines and supports future breeding programs aimed at developing reproductively stable and agronomically superior transgenic cultivars for sustainable crop improvement.

References

1. Chen, Y., Liu, C., Zhang, X., Dong, Y., Yang, J., Wang, D., … & Wang, X. (2025). A Novel Laboratory Protocol for Pollen Viability Assessment to Inform Biosafety Evaluation of Transgenic Rice (Oryza sativa L.). Agriculture, 15(23), 2420.Hu, N., Wang, D., Yuan, Q., Liu, Y., Jiang, H., & Pei, X. (2025). Pollen Quantitative and Genetic Competitiveness of Rice (Oryza sativa L.) and Their Effects on Gene Flow. Plants, 14(13), 1980.Rao, G. S., Deveshwar, P., Sharma, M., Kapoor, S., & Rao, K. V. (2018). Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties. Plant Molecular Biology, 96(1), 35-51.Jia, S., Wang, F., Shi, L., Yuan, Q., Liu, W., Liao, Y., … & Peng, H. (2007). Transgene flow to hybrid rice and its male-sterile lines. Transgenic Research, 16(4), 491-501.Messeguer, J., Marfa, V., Catala, M. M., Guiderdoni, E., & Melé, E. (2004). A field study of pollen-mediated gene flow from Mediterranean GM rice to conventional rice and the red rice weed. Molecular Breeding, 13(1), 103-112.Qu, A., Xu, Y., Yu, X., Si, Q., Xu, X., Liu, C., … & Xu, J. (2021). Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1 (OsGPT1) in rice. Journal of Genetics and Genomics, 48(8), 695-705.Yang, L., Cui, G., Wang, Y., Hao, Y., Du, J., Zhang, H., … & Sun, Y. (2017). Expression of foreign genes demonstrates the effectiveness of pollen-mediated transformation in Zea mays. Frontiers in Plant Science, 8, 383.Singh, S. P., Singh, S. P., Pandey, T., Singh, R. R., & Sawant, S. V. (2015). A novel male sterility-fertility restoration system in plants for hybrid seed production. Scientific Reports, 5(1), 11274.Messeguer, J., Fogher, C., Guiderdoni, E., Marfa, V., Catala, M. M., Baldi, G., & Melé, E. (2001). Field assessments of gene flow from transgenic to cultivated rice (Oryza sativa L.) using a herbicide resistance gene as tracer marker. Theoretical and Applied Genetics, 103(8), 1151-1159.Moon, H. S., Halfhill, M. D., Hudson, L. C., Millwood, R. J., & Stewart Jr, C. N. (2006). Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field. Biotechnology Journal: Healthcare Nutrition Technology, 1(10), 1147-1152.Millwood, Reginald J., Hong S. Moon, Charleson R. Poovaiah, Balasubramaniam Muthukumar, John Hollis Rice, Jason M. Abercrombie, Laura L. Abercrombie, William Derek Green, and Charles Neal Stewart Jr. “Engineered selective plant male sterility through pollen‐specific expression of the Eco RI restriction endonuclease.” Plant biotechnology journal 14, no. 5 (2016): 1281-1290.Zhang, M., Ma, X., Jin, G., Han, D., Xue, J., Du, Y., … & Zhang, X. (2023). A modified method for transient transformation via pollen magnetofection in Lilium germplasm. International Journal of Molecular Sciences, 24(20), 15304.Liu, Y., Ge, F., Liang, Y., Wu, G., & Li, J. (2015). Characterization of competitive interactions in the coexistence of Bt-transgenic and conventional rice. BMC biotechnology, 15(1), 27.Ranjan, R., Khurana, R., Malik, N., Badoni, S., Parida, S. K., Kapoor, S., & Tyagi, A. K. (2017). bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice. Scientific Reports, 7(1), 43397.