Optimized Protocol for in vitro Pollen Germination in Brinjal (Solanum melongena L.)

According to Howlett (1936), the evaluation of pollen viability holds significant importance in the hybridization process. Numerous endogenous factors, such as the nutritional status of the plant, agricultural pesticides and chemicals, can affect the viability of pollen grains in brinjal (Mac Daniels and Hildebrand, 1939; Dubey and Mall, 1972). Pal and Singh (1943) observed that the maximum pollen fertility in brinjal occurs on the second day after flower opening. Popova (1958) further reported that brinjal pollen grains can remain viable for approximately 7-10 days.
 
The success of in vitro pollen germination is influenced by multiple factors, including plant species, nutritional status, timing of harvest, photoperiod, temperature, harvest method, incubation period and the presence of micro and macronutrients in the culture medium (Soares et al., 2008). Adjusting the composition of the culture media for each species is necessary (Chagas et al., 2010; Sinimbu et al., 2011). Carbon sources, boron and other nutrients are essential for the germination of angiosperm pollen (Galleta, 1983). Thompson and Batjer (1950) highlighted that the inclusion of boron in the medium significantly enhances the germination percentage and pollen tube length. Moreover, sugar is commonly used in the culture medium to regulate osmotic balance and provide energy for pollen tube development (Miranda and Clement, 1990).
 
Pollen storage is vital for maintaining viability over time, especially in crossing programs with cultivars having different flowering times. It enables streamlined plantings, eliminates the need for continuous male parent cultivation and maximizes land efficiency. Adjusting temperature, humidity and storage atmosphere extends pollen storage duration. Low temperatures and relative humidity enhance pollen viability. In brinjal hybrid seed production, assessing pollen viability and in vitro germination is crucial. However, designing plant tissue culture media for this purpose is challenging due to complex interactions among factors like sucrose, agar and boron concentrations. The lack of predictability in certain response parameters is often influenced by these key media components and their impact on pollen germination in brinjal.

Plant material
 
The study was conducted in field at Sagdividi Farm, Department of Seed Science and Technology, College of Agriculture, Junagadh Agricultural University, Junagadh during kharif 2020-2021 using randomized block design (Factorial) using parents of brinjal hybrid GJBH 4 and the lab experiment was carried out at Laboratory of Department of Genetics and Plant Breeding, College of Agriculture, Junagadh Agricultural University, Junagadh, with Male parent (JBR 03-16) of hybrid brinjal GJBH 4.
 
Media preparation
 
For in vitro pollen germination, in different eleven solid and liquid media containing different concentration of  sucrose, boron (H3BO3) and Agar  media [1) 2% Sucrose + 4 ppm Boron; 2)  2% Sucrose + 8 ppm Boron; 3) 5% Sucrose + 4 ppm Boron; 4) 5% Sucrose + 8 ppm Boron; 5)  10% Sucrose + 4 ppm Boron; 6) 10% Sucrose + 8 ppm Boron 7) 0.5% Agar + 10% Sucrose + 100 ppm Boron; 8) 1% Agar + 10% Sucrose + 300 ppm Boron 9) 0.5% Agar + 12% Sucrose + 100 ppm Boron; 10) 1% Agar + 12 % Sucrose + 300 ppm Boron 11) Distilled water (Control)] with pH value is 5.8. The range of boron concentration is determined by the physical state of the media. For the first six media, which are in a liquid state, the boron concentration is set within the range of 4 and 8 ppm, while for the remaining four, which are in a solid state, the boron concentration is higher, ranging from 100 and 300 ppm. Basis of fixation of boron concentration depends on solubility and uptake of boron. In liquid media, boron is more readily soluble and accessible to plants or organisms. Therefore, a lower concentration range of 4 and 8 ppm may provide adequate boron for their growth and development. In contrast, solid media may retain boron less efficiently, necessitating a higher concentration range of 100 and 300 ppm to ensure that an adequate amount is available for plant growth. As for the previous steps, solutions were autoclaved for 15 min (121°C), cooled for 30 min, pipetted into 60 mm plastic Petri dishes and stored at 4°C in a refrigerator for use by the next days. The Petri dishes containing pollen were sealed prior and during incubation using a parafilm tape. The pollen germination was in dark for all experiments (used incubators had light bulb but which automatically switched off upon closure of the incubator solid outer door).
 
Flower collection
 
Mature male flowers collected at 8:30-11:00 a.m. Collected flower Keep at room ambient storage condition (~25°C) as well as cold storage condition (refrigerator temp ~3°C).
 
Collection of pollen by extracting anther
 
In vitro germination was carried out immediately followed by twelve hours interval by using of flowers stored under two different condition. Anthers were carefully removed using forceps; the collected anthers are dehisced under sunlight for about 5-10 min, depending upon the intensity of light. Dehisced pollens were subjected to in vitro pollen germination, in different eleven media. A total of 50 anthers were deposited in each Petri dish to ensure availability of sufficient pollen grains for scoring.
 
Pollen incubation
 
The dehisced pollen dusted on petri plate which contain a solidify media and seal the petri dishes with parafilm tape and for liquid media cavity slides (sitting drop technique) is used. Cavity slides are favored for in vitro pollen germination because they provide a controlled environment, simplifying the observation of germination under a microscope. They prevent contamination, ensuring accurate results and are efficient in their use of small sample volumes. The sitting drop culture method is simpler than the hanging drop method. It involves culturing of pollen grains in a drop of culture medium placed on a microslide. The culture is then maintained in a humid chamber to prevent evaporation (Shivanna and Rangaswamy, 1992). This makes them ideal for quick and reliable pollen germination studies. After that incubating the pollen for 3 hours at 25°C.
 
Pollen visualization and count
 
The pollen germination percentage was calculated under stereo-microscope (10 x magnification of Leica Laborlux K microscope with dedicated camera of model Leica EC3) and image capture by MagVision software. Outer growth of the pollen tube beyond the diameter of the pollen was assumed as germinated.
 
Validation of result
 
Female flowers bagged 4 days before hand pollination transfer of anther (stored in two condition) to the female flower using a brush and compare lab germination and fruit setting rate two weeks after pollination. Female parent flowers were enclosed in bags four days before the transfer of anthers of male parent (which were stored in two different conditions). Anthers of male parent were applied to the female flowers using a brush. Two critical outcomes were observed and compared in this experiment. Firstly, the lab germination rate was examined, which measures the success of pollen germination on the stigma and the subsequent growth of pollen tubes to reach the ovules within the female flower. Secondly, the fruit setting rate, or the rate at which successfully pollinated flowers develop into fruits, was assessed. This rate provides insight into the effectiveness of the pollination process in terms of fruit production. The observations and data collection took place over a two-week period following the pollination process. This timeframe was chosen to allow sufficient time for the progression of pollen germination and fruit development. The experiment aims to provide insights into how the storage conditions of anthers and the method of hand pollination using a brush affect the germination and fruit setting rates in female flowers. The percentage fruit setting rate was then calculated as follows:      
 

 
Data analysis
 
The field experiment was conducted using Randomized Block Design (Factorial) repeated thrice as per the method suggested by Cochran and Cox (1957) using the parents of Gujarat Junagadh Brinjal Hybrid 4 (GJBH 4). The observations were recorded on Fruit setting rate (%).Standard germinating medium optimize with Male parent (JBR 03-16) of hybrid brinjal GJBH 4 using completely randomized design with three replications. In vitro pollen germination was carried out with Male parent (JBR 03-16) of hybrid brinjal GJBH 4 at different twelve hours interval of stored pollen. Data on pollen germination were recorded from an average of three repetitions of each treatment. Mean data for pollen germination percentage were analyzed in FCRD (Cochran and Cox, 1957). The observations were recorded on Pollen germination percentage.

Effects of different solid and liquid media on brinjal pollen germination
 
The results presented in Table 1 and Fig 1 and 2 clearly showed that among eleven different treatments (10 media with varied concentrations of sucrose, boron and agar and one control/distilled water), the high germination of fresh pollen was recorded in media containing agar, sucrose and boron (treatment T7 to T10), of which the best germination of fresh pollen (95.00 %) was recorded in treatment T10 (1% Agar + 12% Sucrose + 300 ppm Boron) as shown in Fig 2. A close perusal of the results of in vitro pollen germination (Table 1) showed that the lowest germination percentage was recorded in media with different concentration of sucrose and boron, but not containing agar and there is no pollen germination of taken place in distilled water. The result clearly indicates the importance of sucrose, boron and agar for in vitro pollen germination of brinjal.





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The results are in accordance with the reports of Guler et al., (1995) and Rathod et al., (2018). Guler et al., (1995) reported that most convenient germination medium was 1 per cent agar, 12 per cent sucrose, 300 ppm H3BO3 and 300 ppm Ca(NO3)2 for in vitro pollen germination of eggplant. Rathod et al., (2018) compared three in vitro pollen germination media in Momordica spp. and their interspecific hybrids reported that sucrose @ 15 % + Boric acid @ 0.25% + Calcium nitrite @ 300 mg solution showed the maximum per cent of pollen germination and pollen tube growth. Silva et al., (2020) showed that the agar plays several roles in the germination medium: it promotes the solidification and the osmotic equilibrium of the culture medium, ensures a constant relative humidity and facilitates the incorporation of nutrients, aiding the formation of the pollen tube.
 
Influence of method and period of pollen storage on in vitro pollen germination of brinjal
 
The results presented in Table 2 and Fig 3 revealed that different Method of pollen storage (M) exerted significant effect on Pollen germination (%). Significantly the highest Pollen germination (%) (74.09%) was obtained in the pollen stored in refrigerator compare to ambient condition. These results may be attributed to sufficient loss of pollen viability due to diurnal fluctuations in ambient temperature and relative humidity. Significantly the highest in vitro pollen germination percentage was recorded in <12 hour old pollen (93.50%) and as the duration of pollen storage increased in both the storage conditions, the in vitro pollen germination decreased. These results are in accordance with the findings of Islam and Khan (1998). They collected Bitter gourd pollen samples and stored at four different temperature conditions [room temperature (ranging from 27-30°C), 10°C, 0°C and -5°C for various time periods (0, 3, 6, 9, 12, 15, 25, 35, 45 and 55 days). They recommended that maintaining kakrol pollen grains at low temperatures between 0 and 10 degrees Celsius is effective for preserving them for approximately two months. Khan and Anjum (2006) noticed germination and viability of Solanum melongena L.brinjal pollen for up to 48 weeks. They tested pollen germination using different sucrose and boric acid solutions (ranging from 10% to 100%) and various storage conditions, including refrigeration, freezing, vacuum over silica gel and organic solvents (Acetone, Benzene and Chloroform). They concluded pollen stored at low temperatures (-30°C, -20°C) and freeze-dried pollen (-60°C) exhibited better germination compared to +4°C storage and fresh pollen.





Interaction effect of Method of pollen storage (M) x Period of pollen storage (P) on in vitro pollen germination found significant. Highest pollen germination was observed in case of pollen culture carried out at <12 hour old pollen which was pollen store in refrigerator under lower temperature (95%). Increasing the time interval between pollen collection and culture negatively affected the pollen germination ability.

Influence of method and period of pollen storage on in fruit set (%) of brinjal
 
To assess the accuracy of the in vitro pollen germination in predicting fruit set, Male parent (JBR 03-16) of hybrid brinjal GJBH 4 hand pollinated at different twelve hours interval with stored pollen. The Fruit set (%) exactly followed the same declining trend with the decrease in in vitro pollen germination percentage. Significantly maximum fruit set percentage observed in pollen stored in refrigerator (64.52%) and female flower pollinated with <12 hour old pollen store (84.66%). And the interaction effect found non-significant on fruit set (%) which show in Table 2 and Fig 4.



Our study revealed a positive correlation between the percentage of pollen germination and the fruit set observed in the field after hand pollination. The positive relationship between in vitro germination and in vivo fruit set was also reported by a study on yam (Ng and Daniel, 2000) and other plant species (Volk, 2011).

This study showed that the brinjal pollen germination is primarily influenced by the growing conditions such as the incubation temperature, the medium viscosity and time to use. The best germination of fresh pollen was recorded in treatment of 1% Agar + 12% Sucrose + 300 ppm Boron. A close perusal of the results of in vitro pollen germination showed that the lowest germination percentage was recorded in media with different concentration of sucrose and boron, not containing agar and there is no germination of pollen takes place in distilled water. Significantly the highest in vitro pollen germination and viability percentage was recorded in fresh pollen and as the duration of pollen storage increased in both the storage conditions, the in vitro pollen germination and pollen viability decreased. Of the two storage conditions of pollen, the higher in vitro germination percentage and viability of pollen found in pollen stored in refrigerator compared to ambient storage condition.

All authors declared that there is no conflict of interest.