The emitted light spectrum determines the CRI of light sources and this is then compared against a set of eight standardized color samples. The highest possible CRI is the black body model. Fluorescent light usually range from 50 to 90 CRI.
root cultures of Albizia lebbeck used growth culture medium pH values of 5.0, 5.4, 5.8, 6.2, and 6.6 with pH 5.8 proving to be the preferred level. Further research gathered similar results when working with Azadirachta indica (Indian lilac) and Calophyllum apetalum.
Explain what each of these are. Auxin and cytokine.
Bacterial and fungal contamination of the leaf discs upon the basal and regeneration media significantly impacted the experiment. Viable leaf discs were only obtained through the 20 minute bleach-soak sterilization method. However, 60.92% of the leaf discs were lost to contamination. These results were an improvement over the initial 10 minute bleach-soak protocol which resulted in a 100% loss of leaf disks to contamination. Both sterilization methods utilizing mercuric chloride resulted in the complete loss of viable tissue necessary for in vitro growth.
Contamination of the leaf discs on regeneration media under the different light variables were not statistically significant according to the Student’s T-test (p= 0.20, p= 0.36, p= 0.46). No shoot growth was noted on pH 4 or 8 regeneration media.
African Violet presentation powerpoint 2.0
The Influence of Light Intensity and pH on
Regeneration of African Violet
J. Griffith, T. Wadley, and D.A. Crall
Research Mentor: Dr. Bolyard
African violet, Saintpaulia ionantha, is a leading research model for plant tissue cultures.
Therefore, numerous culturing protocols exist involving consistent growth medium pH
characteristics (5.6-5.8) and lighting intensities. Our team’s undertaking was to manipulate
these two variables by testing five different pH growth media (4.0, 5.0, 6.0, 7.0, 8.0), and three
lights of varying color temperature and Color Rendering Index values (CRI). We hypothesized
the greatest shoot production would result from a growth medium pH of 6.0 due to its
proximity to established pH protocols, 4100K color temperature because it provided a light
spectrum promoting vegetative growth, and 89 CRI due to its light output closest to peak
absorption of chlorophyll. Initial contamination issues were overcome by washing whole
leaves, flaming the leaf punch, and increasing sterilization time from 10 to 20 minutes,
resulting in a 39% reduction in culture contamination loss. All light variables induced shoot
regeneration, however it was only on pH 5, 6 and 7 growth media. Shoot growth on pH 5 and 6
was only under the hypothesized light variable while growth on pH 7 was under the 84
CRI/6500K color temperature value, and 70 CRI/4100K color temperature value.
What is Micropropagation?
• Taking a small tissue culture from a leaf, stem, root, or flower
bud and reproducing exact copies of it on a regeneration
• Takes advantage of the totipotency of plant cells
Why is Micropropagation beneficial?
• Plants exhibiting desired traits such as disease resistance and
crop specific herbicide tolerance can be selected and cultured
for mass production
• Growth and development of medicinal
Exceptional research model
• Able to regenerate by somatic embryogenesis or
• Successful cultures grown from the plant leaves, protoplast,
anther, sub-epidermis, petioles, and flower bud
• Well established growth protocol
• How do light intensity and pH variations effect African violet
• Color rendering index (CRI)
–The capacity of a light source to show the true colors of an
object and a measurement of the accuracy of an illuminant
to an ideal source with the same correlated color
–The higher the CRI value, the higher peak light output near
the red peak absorption of chlorophyll
–References the quality of the colors along the
electromagnetic spectrum and the temperature of a
blackbody radiator that has the same chromaticity of a
particular white light source
–Measured in degrees Kelvin (K)
• The pH of the culture mediums must be within tolerable levels
for the explants to grow successfully
• Little research noted on growth media pH variations with
regards to African violets
• Optimal growth will be observed from a growth media pH of
6.0 due to its proximity to established pH protocols, 4100K
color temperature because it provides a light spectrum
promoting vegetative growth , and 89 CRI due to its light
output closest to peak absorption of chlorophyll.
Materials and Methods
• Media Preparation
– The basal medium used to test leaf disks for contamination contained
30 g/L sucrose and 8 g/L Phytoblend agar.
– Medium used for regeneration contained the 30 g/L sucrose, 8 g/L
agar, 1mM Indole-3-acetic acid (IAA), 1mM zeatin and 4.4 g/L
Murashige and Skoog medium with vitamins.
– Regeneration media was made at varying pH levels (4,5,6,7,8).
Materials and Methods
• Healthy leaves were removed from the African violets and hand washed
with dish detergent for approximately 10 seconds.
• A #6 brass cork borer was heat sterilized before extracting leaf disks.
• Leaf disks were placed in one of three sterilization solutions.
– 10% bleach- 1ml Tween solution for 20 minutes
– 1% mercuric chloride (HgCl2) solution for two minutes
– 0.5% mercuric chloride solution for two minutes
• Sterilized leaf disks were removed and rinsed in 3 separate beakers of
300ml autoclaved water.
• Transfer of all cultures were done using flame-sterilized forceps.
Materials and Methods
• Culture preparation
• Five sterilized discs were placed onto each basal medium plate.
• Plates were placed under lighting of 84 CRI and 6500K color
temperature and monitored for contamination.
• Three to five days later, the leaf disks were transferred via flame-
sterilized forceps, to the varying pH regeneration medium plates and
distributed under the light variables.
• Plates were placed under
the following light variables:
• 89 CRI/4100K (orange
• 84 CRI/6500K (blue light)
• 70 CRI/4100K (green
Materials and Methods
• Plates were examined for contamination daily
• In the event of contamination
• Uncontaminated disks were transferred to new plates,
marked appropriately, sealed with parafilm and returned
to designated lighting
10 MINUTE BLEACH SOAK 20 MINUTE BLEACH SOAK 2 MINUTE 1% HGCL2 SOAK 2 MINUTE 0.5% HGCL2
Effectiveness of Leaf Disc Sterilization Methods. The blue bars represent total discs sterilized
and the orange bars represent remaining viable discs.
Figure 1. Percent of viable leaf discs per pH level. The orange bars
represent the 89 CRI, the blue bars represent the 84 CRI, and the green
bars represent the 70 CRI.
Figure 2. Shoot growth percentage per pH level. The orange bars
represent callus growth under 89 CRI, the blue bar represents callus
growth under 84 CRI, and the green bar represents callus growth
under 70 CRI.
Example of shoot regeneration from a leaf disc plated on pH6
regeneration media under the 89 Color Rendering Index (CRI) and
4100K color temperature value.
• Reevaluation of protocol
resulted in a 39.08%
• 120 leaf discs remained
viable to observe for
Effectiveness of Leaf Disc Sterilization Methods. The blue
bars represent total discs sterilized and the orange bars
represent remaining viable discs.
• Growth only observed from pH levels
5, 6 and 7; however all three light
variables produced shoot
• African violet is capable of growth
under pH levels outside of the ranges
cited in multiple research designs as
well as growth under varying light
• Support of hypothesis
• 30% of the callus growth was
under the orange light (89
• pH 6 growth media accounted
for 20% of the total 30%
Shoot growth percentage per pH level. The orange bars represent
callus growth under 89 CRI, the blue bar represents callus growth
under 84 CRI, and the green bar represents callus growth under 70
• Due to the minute amount of data collected and the short time frame of the
experiment, no conclusive interpretation of the data can be made at this time
• The experiment showed the growing capacity of the African violet was larger than
the research literature suggested.
• Items for consideration in future research
– Thorough examination of the sterilization protocol
– Move the African violet plants to a location away from current
location or divide the plant storage sites into different locations
– Extend leaf washing step to 60 seconds or more
– Use of larger cork borer punch accompanied with extension of the
Bleach-Tween sterilization time
– Partition shelves to keep light for bleeding through to lower levels
– LED lamps vs flourescent
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