The Influence of Light Intensity and pH on Regeneration of African Violet

J. Griffith, T. Wadley, and D.A. Crall Research Mentor: Dr. Bolyard

AbstractAfrican 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 media

• 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

Why the African Violet, Saintpaulia ionantha?

Exceptional research model

• Able to regenerate by somatic embryogenesis or organogenesis

• Successful cultures grown from the plant leaves, protoplast, anther, sub-epidermis, petioles, and flower bud

• Well established growth protocol

Research question

• How do light intensity and pH variations effect African violet regeneration?

Light variables

• 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 temperature (CCT). – The higher the CRI value, the higher peak light output near

the red peak absorption of chlorophyll

Light variables

Color temperature–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)

pH variable

• 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

Hypothesis

• 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 MethodsSurface sterilization• 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.

Material and Methods• Plates were placed under

the following light variables:• 89 CRI/4100K (orange

light)• 84 CRI/6500K (blue light)• 70 CRI/4100K (green

light)

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

Results

10 minute bleach soak 20 minute bleach soak 2 minute 1% HgCl2 soak 2 minute 0.5% HgCl2 soak

0

50

100

150

200

250

300

350

276

307

49

24

0

120

0 0

Num

ber o

f lea

f pun

ches

Effectiveness of Leaf Disc Sterilization Methods. The blue bars represent total discs sterilized and the orange bars represent remaining viable discs.

Results

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.

ResultsExample of shoot regeneration from a leaf disc plated on pH6 regeneration media under the 89 Color Rendering Index (CRI) and 4100K color temperature value.

Discussion• Reevaluation of

protocol resulted in a 39.08% decrease in contamination loss

• 120 leaf discs remained viable to observe for shoot regeneration

10 minute bleach soak

20 minute bleach soak

2 minute 1% HgCl2 soak

2 minute 0.5% HgCl2

soak

0

50

100

150

200

250

300

350

276

307

49

24

0

120

0 0

Num

ber o

f lea

f pun

ches

Effectiveness of Leaf Disc Sterilization Methods. The blue bars represent total discs sterilized and the orange bars represent remaining viable discs.

Discussion• Growth only observed from pH levels

5, 6 and 7; however all three light variables produced shoot regeneration.

• 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 conditions.

• Support of hypothesis• 30% of the callus growth was

under the orange light (89 CRI/4100K)

• 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 CRI.

Discussion• 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.

Discussion

• 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

Literature Cited• Daud, N., Taha, R., & Hasbullah, N. (2008, May 1). Studies on plant regeneration and somaclonal variation in

Saintpaulia ionantha wendl. (African violet). Pakistan Journal of Biological Sciences, pp. 1240-1245.• Duad, N., & Taha, R. (2008). Plant regeneration and floral bud formation from intact floral parts of African Violet

(Saintpaulia ionantha H. Wendle.) cultured in vitro. Pakistan Journal of Biological Sciences, 1055-1058.• Emerson, R, & Arnold, W. (1932). The photochemical reaction in photosynthesis. The Journal of General Physiology,

191-205.• Huan, L. V. T., and Michio Tanaka. "Effects of red and blue light-emitting diodes on callus induction, callus proliferation,

and protocorm-like body formation from callus in Cymbidium orchid." Environment Control in Biology 42 (2004).• Khan, S., Naseeb, S., & Ali, K. (2007). Callus induction, plant regeneration and acclimatization of African violet

(Saintpaulia ionantha) using leaves as explants. Pakistan Journal of Botany, 1263-1268.• Kozai, T., Kubota, C., & Jeong, B. R. (1997). Environmental control for the large scale production of plants through in

vitro techniques. Plant Cell, Tissue and Organ Culture, 49-56.• Lo, K. (1997). Factors affecting shoot organogenesis in leaf disc culture of African violet. Scientia Horticulture, 49-57.• Lo, K., Giles, K., & Sawhney, V. (1997). Acquisition of competence for shoot regeneration in leaf discs of Saintpaulia

ionantha x confusa hybrids (African violet) cultured in vitro. Springer-Verlag.

Literature Cited• Nhut, D. T., Don, N. T., Vu, N. H., Thien, N. Q., Thuy, D. T., Duy, N., & Teixeira da Silva, J. (2006). Advanced

technology in micropropagation of some important plants (Vol. II). United Kingdom: Global Science Books.• Petrus-Vancea, A., & Cachita-Cosmo, D. (2008). Biochemical determinations made on African violets (Saintpaulia

ionantha) exvitroplantlets, being illuminated during their acclimatization to a septic medium, with different types of light. Studia Universitatis "Vasile Goldis", Seria Stintele Vietii (Life Sciences Series), 81-86.

• Polking, G., & Stephans, L. (1995). Plant micropropagation using african violet leaves. • Rout, G., Mohapatra, A., & Mohan Jain, S. (2006). Tissue culture of ornamental pot plant: A critical review on

present scenario and future prospects. Biotechnology Advances, 531-560.• Schubert, E. F., & Kim, J. K. (2005). Solid-State Light Sources Getting Smart. Science, 1274-1278.• Seibert, M., Wetherbee, P. J., & Job, D. D. (1975). The effects of light intensity and spectral quality on growth and

shoot initiation in tobacco callus. Plant Physiology, 130-139.• Sunpui, W., & Kanchanapoom, K. (2002). Plant regeneration from petiole and leaf of African violet (Saintpaulia

ionantha Wendl.) cultured in vitro. The Songklanakarin Journal of Science and Technology , 357-364.• Taha, R. M., Duad, N., & Hasbullah, N. A. (n.d.). Establishment of efficient regeneration system, acclimatization

and somaclonal variation in Saintpaulia ionantha H. wendle.