30 May 2014

Jennifer Griffith255 Grooms RdBruceton, TN 38317678/449-6008; 731/586-7209Email: Jennifer.griffith@my.uu.edu

Research Mentor: Dr. Mark Bolyard

THE INFLUENCE OF LIGHT INTENSITY AND PH ON REGENERATION OF AFRICAN VIOLET, SAINTPAULIA IONANTHA

Jennifer Griffith, Taylor Wadley, and Daniel Crall, Department of Biology, Union University,

1050 Union University Dr, Jackson, TN 38305

Abstract: The African violet (Saintpaulia ionantha) is a leading research model for plant

tissue cultures. Therefore, numerous culturing protocols exist concerning regeneration

media pH characteristics (5.6-5.8) and lighting intensities. Our team’s undertaking is to

manipulate these two variables by testing five different pH growth media levels (4.0, 5.0,

6.0, 7.0, 8.0), and three lights of varying color temperature and color rendering index (CRI)

values. We hypothesize 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

peak light output near the red peak absorption of chlorophyll which is 700 nm.

INTRODUCTION

Micropropagation has become a massive area of research and development in the

commercial plant propagation industry, as well as in the scientific community. Both groups have

been involved in finding new techniques and procedures to improve the quality and quantity of

plant production. Specific attention is being focused on organogenesis, survival rate, growth and

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development of medicinal compounds (Duad et al. 2008; Khan et al. 2007; Malik et al. 2012;

Nhut et al. 2006; Rout et al. 2006). Furthermore, plants expressing desired traits, such as a

specific disease resistance or crop specific herbicide tolerance, can be selected and mass

produced through micropropagation techniques. This ensures that high quality plants and fruit

will be produced which cannot always be maintained through the heterozygous reproduction

obtained through seeds. Instead, micropropagation is a dependable technique to obtain

genetically pure populations through in vitro propagation. Other advantages of micropropagation

are that large numbers of plants can be raised from small explants in a short time, it is

economical and reliable to produce disease free plants, it is not limited to seasonal growing

seasons, and stocks can be maintained for years (Malik et al. 2012).

African violets are native to eastern tropical Africa and have gained popularity in

America due to its small size, ornamental appeal, ability to grow under artificial light, and shade

tolerance (Lo 1997; Nhut et al. 2006). However, its ability to regenerate by somatic

embryogenesis or organogenesis has established it as an important research model (Taha et al.

2010). Furthermore, researchers have successfully grown cultures taken from the plant leaves,

protoplast, anther, sub-epidermis, petioles, and flower buds (Duad and Taha 2008; Khan et al.

2007; Taha et al. 2010). They also have illustrated the outcomes of in vitro growth when

experimental variables such as growth factors, medium type, leaf disc orientation on medium,

light intensity variations, leaf age at culture, and wounds on leaf disc where manipulated and

studied (Duad and Taha 2008; Khan et al. 2007; Lo 1997; Lo et al. 1997; Nhut et al. 2006;

Sunpui and Kanchanapoom 2002).

The purpose of this experiment is to manipulate light intensity, as well as growth medium

pH, in order to determine the effects of these two variables on in vitro growth of African violets.

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Light intensity variables specific to this experiment are color temperature and CRI values. The

second experimental variable, pH, has been less studied. The pH of the culture mediums must be

within tolerable levels for the explants to grow and process nutrients at the molecular level. We

will be looking to see if light intensities and pH variations collectively alter African violet tissue

cultures. Three different fluorescent light sources, consisting of 89 Color Rendering Index (CRI)

and 4100K color temperature value, 84 CRI and 6500K color temperature value, and 70 CRI

with 4100K color temperature values are to be used and the pH of growth medium is to be 4.0,

5.0, 6.0, 7.0, and 8.0. Samples will be sterilized, cultured and kept at room temperature with a 16

hour light time and 8 hour dark time. We hypothesized the best growth 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.

LITERATURE REVIEW

Organogenesis/Totipotency

The African violet, from the family Gesneriaceae, is a popular commercial houseplant due to its

variety of colors and shapes, ability to thrive indoors without direct sunlight and artificial light,

and the ease to propagate new offspring all year (Sunpui and Kanchanapoom 2002).

Micropropagation, or in vitro organogenesis, specifically involves adventitious organ formation

from explants with active cell division. These adventitious organs arise in two ways; directly

from the original explant tissue (direct organogenesis) or indirectly through a callus (indirect

organogenesis) (Taha et al. 2010). New plant cells have the ability to become any possible cell in

that is needed in the plant. This is an example of totipotency. Totipotency is when a single tissue

sample, as in a piece of a leaf or stem, can be used to regenerate a new plant if provided with the

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appropriate growing conditions. All the necessary genetic information for growth is contained

within the DNA of the cells in a given tissue. The cells are de-differentiated and then re-

differentiated to become a new plant identical to the original parent. From this cells form

unipolar structures through organogenesis, or undergo somatic embryogenesis and form bipolar

structures. These new structures occur either directly on the explant or on callus formation which

was initially formed on the explant (Rout et al. 2006). Lo (1997a) showed that older leaf tissues

had decreased organogenic ability, but leaves in certain developmental stages had higher

regenerative capacities than the oldest or youngest. Additionally, in vitro regeneration follows

three developmental events; acquisition of competence, induction, and determination.

Acquisition of competence occurs when the explant can be successfully grown on callus-, root-,

or shoot-inducing medium. Induction occurs next when growth regulators in the medium cause

the explant to develop along a specific developmental pathway. Determination starts when there

is continued growth along a certain pathway even after the removal of growth regulators (Lo et

al. 1997).

Lack of cellular competence is one of the major blocks to in vitro regeneration. Studies

have shown that there is a “window” of competence to which cells can be induced, and afterward

will no longer be competent. In S. ionantha x confusa hybrids cultures were not competent in for

the first three days (Lo et al. 1997). Inducing medium contains water, sugar, agar, macro-

elements, trace elements, vitamins, and various growth hormones which are a combination of

auxins and cytokinins. Variations in hormone levels and types of hormones will cause induction

of callus formation or production of roots and sprouts (Harclerode 1979). Cytokinins will induce

shoot formation for most plants, and the addition of auxins is essential for shoot induction and

multiplication. High concentrations of cytokinins will fail to produce shoot formations from

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leaves or petiole explants, but low concentration will cause high rates of shoot bud regeneration.

The amount and type of auxins and cytokinins are species specific, and are not always well

known (Rout et al. 2006). One of the determining factors of patterns of morphogenesis from

petiole explants to increase shoot number is the auxin/cytokinin balance. Change in the balance

and combination of these hormones can alter morphogenetic responses. African violets have

shown that leaf explants do not seem to have a low auxin requirement for callus growth (Sunpui

and Kanchanapoom 2002). Khan et al. (2007) tested three different auxins for their percentage

of callus induction and showed that napthyl acetic acid (NAA) produced a higher percentage

than indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA). Taha et al. (2010) and Daud et al.

(2008) showed that 1.0 mg/L IAA and 2.0 mg/L zeatin in Murashige and Skoog (MS) medium

was well suited for shoot regeneration over NAA and 6-benzylaminopurine (BAP) and produced

more shoots and roots and 100% of the time.

Contaminants

Critical to the success of the project, will be to prevent or avoid microbial contamination

of the plant tissue cultures. Contaminates may originate from the laboratory environment,

researchers, mites and thrips, ineffective sterilization techniques, or from the plants themselves.

Aspectic techniques will control microorganisms that might be introduced to cultures or can be

found on the surface of cultures. Using autoclaves and laminar flow hoods are the first steps to

avoid environment contaminants found in the lab. The hardest to detect will be those that are

found within the plant tissues themselves. Most of these are due to endophytic microbes

associated with soil or water found in cell junctions and intercellular spaces of cortical

parenchyma. Use of filtered water is suggested to reduce some of the bacterial contaminates.

Fresh mixtures of disinfectants are also advised due possible loss of strength of the disinfectant.

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(Reed and Tanprasert 1995). Surface sterilization of the tissue cultures an important procedure to

be performed before placing cultures on medium. Khan et al. (2007a) described washing the

leaves in running tap water for 10 minutes to remove surface particulates. Ahmed et al. (2012)

used a diluted solution of sodium hypochlorite (commercial bleach) and a few drops of Tween

20 to disinfect plant tissues. Sodium hypochlorite is used as the disinfectant in concentration

between 5-50% and Tween 20 as an emulsifier. Lo et al. (1997b) used a 10% sodium

hypochlorite for 20 mins with success. Taha et al. (2010) also used three rinses with sterile

distilled water after soaking tissue cultures in disinfectant.

Light

One of the most important abiotic factors to establish and develop plant cultures is the

use of light. Light is used as the energy source for photosynthetic organs. It is a fundamental

environmental cue to a plant’s life by directly and indirectly affecting the regulation of

development and growth (Nahar et al. 2012). Plants grown in low-light have been shown to be

more susceptible to photoinhibition than those grown under high-light intensity. This shows that

there is a correlation between increased light intensity and net photosynthesis rate, but if too high

of intensity is reached it will also decrease the net photosynthesis rate. If a plants photosynthetic

apparatus cannot dissipate excessive light energy quickly enough photosynthetic efficiency is

reduced and damage to the photosynthetic reaction center (Fan et al. 2013). The reaction to

different lighting conditions is species specific and also varies during growth stages. The quality

and wavelength of light can influence different types of development (Rout et al. 2006). Several

plant anatomical, physiological, morphological, and biochemical parameters can be changed due

to variations in light quality.

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The three major types of information-transducing photoreceptors are phytochromes, blue-

light receptors, and UV-B photoreceptors. Green-light-mediated responses might also be

received by zeaxanthin-based compounds, but this is still speculated. Phytochromes are

photointerconvertible, using red and far-red light, soluble pigmented proteins. These

photoreceptors are responsible for germination, seedling establishment, flowering, dormancy,

nyctinasty, stomatal development, plant architecture, and shade avoidance. Blue-light receptors,

such as cryptochrome family, are involved in light-signal transduction regulating phototropism,

de-etiolation, chloroplast movements, light-induced stomatal opening, photoperiod-dependent

flowering induction, and resetting circadian oscillator. The other major class of blue-light

receptors are phototropins which optimize photosynthesis by phototropism, chloroplast

movements, and stomatal opening. The blue-green light receptor and UV-B also controls

stomatal opening along with the other photoreceptors (Macedo et al. 2010). Plant leaves

absorbed approximately 90% of available blue or red light, and the absence of one or the other

causes photosynthetic inefficiencies (Fan et al. 2013). Normal cool-white fluorescent lamps

provide blue, yellow, and green light but do not produce much red light. Low-energy plants,

which are most houseplants, grow better with indirect light and lower intensity light. They

require about 15 lamps watts per square foot. High-energy plants require more far-red light and

need about 20 lamp watts per square foot (Osram Sylvania 2000). Red (660 nm), white (400

nm), blue (430 nm), yellow (580 nm) and green (544 nm) are the wavelengths that have been

shown to improve growth of various types. Red has been shown to increase shoot and root

growth (Rout et al. 2006; Petrus-Vancae and Cachita-Cosma 2008). Some studies suggest that a

combination of blue and red light will give the highest quality plants cultured in vitro (Macedo et

al. 2010). Successful parameters used in previous research include using a 13−70 µmol m−2 s−1

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light intensity range, also known as photosynthetic photon flux density (PPFD) (Khan et al.

2007; Lo 1997; Lo et al. 1997; Nhut et al. 2006; Sunpui and Kanchanapoom 2002). Fluorescent

lamps are used due to their ability to have a relatively uniform horizontal PPFD over an entire

shelf of cultures and they have a spectrum that will generally match the requirements for in

vintro propagation (Kozai et al. 1997). Researches inducing regeneration from petioles and floral

bud used 13-20 PPFD (Duad and Taha 2008; Sunpui and Kanchanapoom 2002). While those

inducing regeneration from leaves, used 70 PPFD (Lo 1997; Lo et al. 1997). There are many

discrepancies in literature though as to what wavelengths truly improves or inhibits growth.

Color temperature, measured in degrees Kelvin, refers to the light quality coming out of

the light source. This 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 (Schubert and Kim 2005). It is important to plants because a higher color temperature

promotes floral growth while a lower value promotes vegetative growth. CRI uses the

trichromatic design of the human visual system. It is the capacity of a light source to show the

true colors of an object (Schubert and Kim 2005) and a measurement of the accuracy of an

illuminant to an ideal source with the same correlated color temperature (CCT). 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. This is important because the higher the number, the

higher peak light output near the red peak absorption of chlorophyll (Taiz and Zeiger 2010).

Emerson and Arnold found that Chlorella pyrenoidosa cells had different amounts of chlorophyll

per unit amount of cells based on the type of light under which they were grown. The

concentration of chlorophyll, along with the Blackman reaction development, is what drives

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photosynthesis for plants (Lee et al. 1985; Schubert and Kim 2005). Light is an important

environmental cue in the life cycle of plants, and regulated development and growth both

directly and indirectly (Cybularz-Urban et al. 2007). When looking at callus growth in Cymbidiu

orchid cultures, green light sources propagated increased numbers of callus tissues over white,

red, or blue (Nahar et al. 2004).

Growth Media pH

Very little research has been performed to show the possible tolerance ranges for pH of

medium with the African violet. It has been generally assumed that a pH of 5.6-5.8 for growth

media has the best (Khan et al. 2007; Lo 1997; Lo et al. 1997; Nhut et al. 2006; Sunpui and

Kanchanapoom 2002). Skirvin et al. (1986) states that most tissue cultures are able to tolerate

values between 5.2 and 5.8. They also showed that higher pH levels, particularly between 5.7 to

8.5, have significant differences between initial pH levels and pH levels after autoclaving.

Research conducted on 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 (Perveen et al. 2011).

Further research gathered similar results when working with Azadirachta indica and

Calophyllum apetalum.

MATERIALS AND METHODS

Media Preparation

The basal media that will be used to test leaves for contamination will contain 30 g/L

sucrose and 8 g/L Phytoblend agar. The medium will be made in a 1 L Erlenmeyer flasks with

the volume filled to 500 mL. The flasks will need to be autoclaved for 15 minutes. Once cooled

to room temperature the medium is to be poured into the petri dishes and stored in a designated

laboratory refrigerator for later use. The media that will be used for regeneration will contain 30

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g/L sucrose, 8 g/L agar, 1mM Indo-3-acetic acid (IAA), 1mM zeatin and 4.4 g/L Murashige and

Skoog medium. The regeneration media will be made of varying pH levels (4,5,6,7,8). The pH

will be adjusted with 0.5 M sodium hydroxide and 2.5 M hydrochloric acid.

Surface sterilization

Healthy leaves are to be removed via scalpel blade from seven African violet plants kept in the

laboratory. Leaves will need to be hand washed with dish detergent in a large beaker within the

sink for at least 1 min, rinsed thoroughly with running tap water and placed on a clean sheet of

aluminum foil. A #6 brass cork borer (punch) is to be used to obtain culture samples. The punch

will be washed with hand soap prior to use for 10 seconds, dried with paper towels, the tip

flamed over a Bunsen burner, allowed to cool and used to extract leaf discs. Discs will be

gathered in sets of 25 or 50, wrapped inside a clean sheet of aluminum foil and transported to the

EdgeGARD® plant tissue culture hood. The work station is to be cleaned prior to, and

immediately following, all work performed by spraying the working surface with a 75% ethyl

alcohol solution and wiped thoroughly with paper towels. Cultures are to be removed from the

foil and placed in a 10% Bleach-Tween solution (30 ml bleach,1 ml Tween, 270 ml dH2O). They

will need to remain in the solution for 20 minutes before being removed and placed in a beaker

of 300 ml autoclaved water. After a one minute rinse they will be transferred to a second beaker

of autoclaved water. This will need to be repeated once more. Transfer of all cultures were

performed using flame-sterilized forceps. After the third rinse, the cultures were placed onto the

regeneration media. A second form of sterilization was utilized for comparison to the bleach

sterilization method. A 1% mercuric chloride solution (3 ml HgCl2, 297ml dH2O) was prepared

and leaf discs added to it for a total soak time of two minutes before being removed and placed

in a beaker of 300 ml autoclaved water. After one minute they were transferred to a second

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beaker of autoclaved water. This was repeated once more. Transfer of all cultures are to be

performed using flame-sterilized forceps. After the third rinse, the cultures will be placed onto

the regeneration media in the same manner as the bleach sterilization technique.

Culture Preparation

All cultures will be transfered using flame-sterilized forceps. Five individual leaf discs

are to be placed onto each basal media plate. Each basal media plate is to contain only 30 g/L

sucrose and 8 g/L agar, no growth hormones are to be used until disc have been confirmed to be

free of contamination. Each plate is to be labeled with the date made, sterilization method used

on leaf discs (Bleach or HgCl2), and experiment name. A strip of parafilm will need to be

secured over the entirety of the plate to ensure a complete seal from outside contamination.

Plates are to be placed under lighting of 84 CRI and 6500K color temperature (blue light) and

monitored for contamination. Three to five days later, the plates will need to be brought to the

EdgeGARD® plant tissue culture hood where the leaf discs can be transferred via flame-

sterilized forceps, to the varying pH regeneration media plates containing 30 g/L sucrose, 8 g/L

agar, 1 mM Indo-3-acetic acid (IAA), 1 mM zeatin and 4.4 g/L Murashige and Skoog. The plate

will be labeled with the date made, pH of plate, sterilization type and light variable to be placed

under; either 89 CRI and 4100K (orange light), 84 CRI and 6500K (blue light), or 70 CRI with

4100K (green light) color temperature value. A strip of parafilm is to be secured over the entirety

of the plate to ensure a complete seal from outside contamination. In the event of contamination

of either plate types (basal media or regeneration media), plates are to be brought to the

EdgeGARD® plant tissue culture hood and uncontaminated discs will be transferred to new

plates, marked appropriately, sealed with parafilm and returned to designated lighting. All

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personnel is to wear gloves when cleaning, sterilizing, and transferring leaf discs in order to

reduce exposure to contamination.

Lighting

The fluorescent lights will be suspended from two metal shelving units. There will be

three shelves each of bulbs rated 89 CRI/4100K (designated orange light), 84 CRI/6500K

(designated blue light), and 70 CRI/4100K (designated green light). All lights are to be plugged

into a main outlet strip and attached to a timer set to 16 hours on, 8 hours off.

EQUIPMENT LIST

7 potted African violet plants - $25

~200 Petri dishes

8 g/L Phytoblend agar

30 g/L Sucrose

3 Erlenmeyer flasks

6 large beakers

1mM Indo-3-acetic acid (IAA)

1mM zeatin

4.4 g/L Murashige and Skoog medium

0.5 M sodium hydroxide

2.5 M hydrochloric acid

Microwave

Autoclave

Autoclave tape

#6 brass cork borer - $25

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Inoculation loop

Scalpel

5 metal tweezers

Dishwater soap

Bleach

Bunsen burner

Aluminum foil

Parafilm

EdgeGARD® plant tissue culture hood

75% ethyl alcohol

Tween 20

mercuric chloride

Fluorescent bulbs (12 of each): 89 CRI/4100K, 84 CRI/6500K. and 70 CRI/4100K - $290

2 metal shelving units

Power outlet strip - $10

Light timer - $10

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Cybularz-Urban, Teresa, Ewa Hanus-Fajerska, and Adam Swiderski. 2007. Effect of light

wavelength on in vitro organogenesis of a Cattleya hybrid. ACTA Biologica

Cracoviensia Series Botanica 49/1:113-118.

Daud, Norhayati, Rosna Mat Taha, and Nor Azlina Hasbullah. 2008a. Studies on plant

regeneration and somaclonal variation in Saintpaulia ionantha wendl. (African violet).

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Duad, N., and R. M. Taha. 2008b. Plant Regeneration and floral bud formation from intact floral

parts of African Violet (Saintpaulia ionantha H. Wendle.) cultured in vitro. Pakistan

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Fan, Xiao-Xue, Zhi-Gang Xu, Xiao-Ying Liu, Can-Ming Tang, Li-Wen Wang, and Xue-lin Han.

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