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, KY
Figure 6. C. retusa percent pollen germination after buffered KAuCl4 treatment
Results
Abstract
100
Figure 3. C. retusa pollen germination and tube length after KAuCl4 treatment
80
Percent Germination
Unique 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 dosagedependent 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 dosage–dependent effects of buffered KAuCl4-supplemented
GM on pollen germination and tube length was largely comparable with those of the non-buffered KAuCl4supplemented 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.
60
40
20
10 ppm
50 ppm
100 ppm
0
0 ppm 10 ppm 25 ppm 50 ppm 100 ppm 250 ppm 500 ppm 1000
ppm
KAuCl4 [ppm]
KAuCl4 (ppm)
Figure 7. C. retusa pollen tube length after buffered KAuCl4 treatment
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.
1000 ppm
200
160
Pollen tube length (uM)
Nanotechnology 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).
500 ppm
Figure 4. C. retusa percent pollen germination after non-buffered KAuCl4 treatment
80
60
40
120
80
40
Percent Germination
Introduction
250 ppm
20
0
0
0 ppm 10 ppm 25 ppm 50 ppm 100 ppm 250 ppm 500 ppm 1000
ppm
-20
KAuCl4 [ppm]
0 ppm 10 ppm 25 ppm 50 ppm 100 ppm250 ppm500 ppm 1000
ppm
KAuCl4 [ppm]
KAuCl4 (ppm)
-40
Materials and Methods
-60
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).
-80
Figure 5. C. retusa pollen tube length after non-buffered KAuCl4 treatment
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.
Figure 1. Crotalaria retusa
plant
Figure 2. Crotalaria retusa
flower
240
•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 nonbuffered solutions.
Acknowledgements
200
Pollen tube length (uM)
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.
Conclusion
KAuCl4 (ppm)
This research was supported by the NSF HBCU-UP grant awarded to Sarwan K. Dhir at the
Fort Valley State University, Fort Valley, GA.
125
160 mg/L
3000 mg/L
120
80
40
5000 mg/L
0
0 ppm 10 ppm 25 ppm 50 ppm 100 ppm250 ppm500 ppm 1000
ppm
KAuCl4 [ppm]
KAuCl4 (ppm)
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