Dosage Dependent Affect of KAuCl4 on the Pollen Germination of Crotalaria retusa

Shandrea Stallworth, Center for Biotechnology, Fort Valley State University, Fort Valley, GA Ajay Jain and Shivendra V. Sahi, Biotechnology Center, Department of Biology, Western Kentucky University, Bowling Green, KYUnique catalytic properties of gold nanoparticles (AuNPs) could be achieved by manipulating their geometries. Recent studies have shown the in planta synthesis of AuNPs and manipulation of their geometries in the roots of plant species. However, due to the lignified cell wall of the plant tissue, isolation of biomatrix-embedded AuNPs have not been met with any success so far. On the contrary, in vitro germinated pollen tubes have been successfully used for the isolation of sperm nuclei by subjecting them to osmotic shock. Several studies have used pollen systems for dosage-dependent affects of various toxic heavy metals. However, the effect of KAuCl4 on the in vitro germination and pollen tube length is not known. Crotalaria retusa pollen shows high percent germination and is amenable for long-term storage. C. retusa pollen was used to decipher the dosage-dependent (0 - 1000 ppm) effects of KAuCl4 on percent pollen germination and tube length. Interestingly, compared with the control (0 ppm) there were significant increases in both percent germination and pollen tube length upon supplementing the germination medium (GM) with 10 ppm of KAuCl4. When GM is supplemented with > 100 ppm of KAuCl4 it induced significant reductions in percent germination and pollen tube length. This study clearly demonstrates the hormesis effect of gold treatment on pollen germination and tube length. However, supplementation of GM with increasing concentrations of KAuCl4 resulted in a significant decline in the pH values ranging from 6.25 (0 ppm) to 2.56 (1000 ppm). To further determine whether the observed effect was due to KAuCl4 or because of the resultant shift in the pH, KAuCl4-supplemented GM was buffered to pH 6.0 with MES buffer. Interestingly, the dosagedependent effects of buffered KAuCl4-supplemented GM on pollen germination and tube length was largely comparable with those of the non-buffered KAuCl4-supplemented GM. Efforts are now underway to use the mass pollen culture for the synthesis of AuNPs that would be confirmed by Transmission Electron Microscopy and Energy Dispersive X-ray Spectroscopy.

KAuCl4, in non-buffered solutions, shows a stimulatory effect on percent pollen germination and tube growth at 10 ppm. The hormesis effect was observed and there was a steady decline in germination and pollen tube growth at dosages of 25 ppm or more. When KAuCl4 solutions were buffered, the same effect was observed but it was not as dramatic as the non-buffered solutions. This research was supported by the NSF HBCU-UP grant awarded to Sarwan K. Dhir at the Fort Valley State University, Fort Valley, GA.1. Ratner, M.; Ratner, D. Nanotechnology: A Gentle Introduction to the Next Big Idea; Prentice Hall: Upper Saddle River, NJ, 2003.2. Sharma, N.C.; Sahi, S.V.; Nath, S.; Parsons, J.G.; Gardea-Torresdey, J.L.; Pal, T.; Synthesis of Plant Mediated Gold Nanoparticles and Catalytic Role of Biomatrix-Embedded Nanonomaterials. Environmental Science and Technology. 2007, 41, 5137-5142.3. Starnes, D.L.; Jain, A.; Sahi, S.V.; In-Planta Engineering of Gold Nanoparticles of Desirable Geometries by Fine-tuning Growth Conditions: An Eco-friendly Approach. Environmental Science and Technology. 2010. In Press.4. Shivanna, K.R.; Xu, H.; Taylor, P.; Knox, R.B.; Isolation of Sperms from the Pollen Tubes of Flowering Plants During Fertilization. Plant Physiology. 1988, 3, 647-650. 5. Jain, A.; Shivanna, K. R. Loss of Viability During Storage is Associated with Changes in Membrane Phospholipids. Phytochemistry. 1989, 28, 4, 999-1002.6. Gass, N.; Glagotskaia, T.; Mellema, S.; Stuurman, J.; Barone, M.; Mandel, T.; Roessner-Tunali, U.; Kuhlemeier, C.; Pyruvate Decarboxylase Provides Growing Pollen Tubes with a Competitive Advantage in Petunia. Plant Cell. 2005, 17, 2355-2368.5000 mg/L125 mg/L3000 mg/LFigure 3. C. retusa pollen germination and tube length after KAuCl4 treatmentFigure 4. C. retusa percent pollen germination after non-buffered KAuCl4 treatmentFigure 5. C. retusa pollen tube length after non-buffered KAuCl4 treatmentFigure 6. C. retusa percent pollen germination after buffered KAuCl4 treatmentFigure 7. C. retusa pollen tube length after buffered KAuCl4 treatmentKAuCl4 (ppm)KAuCl4 (ppm)AbstractIntroductionNanotechnology is a fast growing field of science that deals with nanomaterials ranging from 1-100 nanometers. Nanogold particles (AuNPs) have found applications in medicine, consumer goods, heavy industry, information and communication, optoelectronic devices, environment-friendly energy systems, chemical catalysis, and the list continues to grow (1). Although wet synthesis is traditionally used for the synthesis of AuNPs, the process generates toxic byproducts that have adverse environmental implications. In planta synthesis of AuNPs is a viable alternative and recent study has demonstrated the feasibility of generating AuNPs of desirable geometries by manipulating growth conditions (2, 3). However, extraction of the biomatrix-embedded AuNPs has not been met with any success so far largely due to the difficulty in the dissolution of lignified roots. Unlike roots, pollen tubes are largely made of pectin and sperm nuclei have been isolated by mere osmotic shock (4). Although several studies have used in vitro germination of pollen grains for testing the toxicity effects of various heavy metals like Hg, Cd, Pb etc., it is not known if they can be used for the synthesis of AuNPs. For testing the feasibility of using pollen for the synthesis of AuNPs, it is desirable to identify a plant species that produces large amount of pollen grains that could be easily stored without adversely affecting the percent germination and pollen tube growth. Crotalaria retusa are amenable for long term storage and show high in vitro percent germination (5).

In present study the dosage dependent affect of KAuCl4 on percent germination and tube length of C. retusa was investigated. This study was aimed to identify an optimal KAuCl4 concentration that does not have an adverse affect on both the percent germination and pollen tube growth with a long-term objective of using this optimal KAuCl4 concentration for the subsequent synthesis of AuNPs in mass-cultured in vitro germinated pollen tubes.Plant Material. Flowers were collected from C. retusa grown in the greenhouse of Western Kentucky University. Pollen was collected from freshly dehisced anthers and was used immediately (4).

Germination Media. Germination media was prepared in a 5x concentration from Petunia hybrida germination media without polyethylene glycol and buffered to pH 5.8 with 0.1M MES buffer (6).

KAuCl4 Concentrations. Sixteen 15 ml falcon tubes were filled with 10 ml of germination media. KAuCl4 concentrations were made from 1mg/mL (0-100 ppm) and 50mg/mL (250-1000 ppm) stocks. Eight tubes were left non-buffered while the other eight were buffered to pH 6.0 with 0.1M MES buffer.

Pollen Germination and Pollen Tube Growth Observation. Using a camel hair brush, freshly collected pollen was spread onto microscope slides. 20 uL of 0 ppm non-buffered germination media was added to three of the slides and mixed together. After being properly mixed, another 20 uL of germination media was added to each slide. The slides were then placed in a humidity chamber for three hours. This was repeated for 10, 25, 50, 100, 250, 500, and 1000 ppm. After three hours all reactions were stopped with ethanol: lactic acid (2:1). Observations were measured with a light microscope. The experiment was then repeated with the buffered KAuCl4 solutions. Materials and MethodsResultsConclusionAcknowledgementsReferences