Introduction to Research Proposal
- 1. 30 May 2014 Jennifer Griffith 255 Grooms Rd Bruceton, TN 38317 678/449-6008; 731/586-7209 Email: 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
- 2. Griffith 2 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.
- 3. Griffith 3 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
- 4. Griffith 4 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
- 5. Griffith 5 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.
- 6. Griffith 6 (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.
- 7. Griffith 7 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 −
- 8. Griffith 8 70µmol m−2 𝑠−1 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
- 9. Griffith 9 concentration of chlorophyll, along with the Blackman reaction development, is what drives 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
- 10. Griffith 10 laboratory refrigerator for later use. The media that will be used for regeneration will contain 30 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
- 11. Griffith 11 in a beaker of 300 ml autoclaved water. After one minute they were transferred to a second 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
- 12. Griffith 12 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
- 13. Griffith 13 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 LITERATURE CITED Ahmed , A. Bakrudeen Ali, S. Mohajer, E.M. Elnaiem and R.M. Taha 2012. In vitro Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants. Available at: http://www.intechopen.com/books/plant-science/in-vitro-regeneration- acclimatization-and-antimicrobial-studies-of-selected-ornamental-plants
- 14. Griffith 14 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). Pakistan Journal of Biological Sciences 11(9):1240-1245. 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 Journal of Biological Sciences 11(7):1055-1058. Fan, Xiao-Xue, Zhi-Gang Xu, Xiao-Ying Liu, Can-Ming Tang, Li-Wen Wang, and Xue-lin Han. 2013. Effects of light intensity on the growth and leaf development of young tomato plants grown under a combination of red and blue light. Scientia Horticulturae 153:50-55. Harclerode, John B. 1979. Affects of rooting hormones on African violet cuttings (Saintpaulia ionantha). Thesis Submitted to Department of Biology Emporia State University, Emporia, Kansas. Kataky, A., and P. J. Handique. 2010. Micropropagation and screening of antioxidant potential of Andrographis paniculata (Burm. f) Nees. Journal of Hill Agriculture 1(1):13-18. Khan, Saifullah, Saima Naseeb, and Kashif Ali. 2007. Callus induction, plant regeneration and acclimatization of African violet (Saintpaulia ionantha) using leaves as explants. Pakistan Journal of Botany 39(4):1263-1268.
- 15. Griffith 15 Kozai, Toyoki, Chieri Kubota, and Byoung Ryoung Jeong. 1997. Environmental control for the large-scale production of plants through in vitro techniques. Plant Cell, Tissue and Organ Culture 51:49-56. Lee, Ni, Hazel Y. Wetzstein, and Harry E. Sommer. 1985. Effects of quantum flux density on photosynthesis and chloroplast ultrastructure in tissue-cultured plantlets and seedlings of Liquidambar styraciflua L. towards improved acclimatization and field survival. Plant Physiol 78:637-641. Leifert, C., W. M. Waites, and J. R. Nicholas. 1989. Bacterial contaminants of micropropagated plant cultures. Journal of Applied Bacteriology 67:353-361. Lo, K. H. 1997a. Factors affecting shoot organogenesis in leaf disc culture of African violet. Scientia Horticulture 72:49-57. Lo, K. H., K. L. Giles, and V. K. Sawhney. 1997b. Acquisition of competence for shoot regeneration in leaf discs of Saintpaulia ionantha x confusa hybrids (African violet) cultured in vitro. Plant Cell Reports 16:416-420. Macedo, Andrea Furtado, Marcos Vinicius Leal-Costa, Eliana Schwartz Tavares, Celso Luiz Lage, and Maria Apparecida Esquibel. 2011. The effect of light quality on leaf production and development of in vitro-cultured plants of Alternanthera brasiliana Kuntze. Environmental and Experimental Botany 70:43-50. Malik, C. P., Poonam Garg, Yaksha Singh, and Staffi Grover. 2012. Medicinal uses, chemical constituents and micropropagation of three potential medicinal plants. International Journal of Life science & Pharma Research 2:57-76.
- 16. Griffith 16 Nahar, S. J., K. Shimasaki, and S. M. Haque. 2012. Effect of different light and two polysaccharides on the proliferation of protocorm-like bodies of Cymbidium cultured in vitro. VII International Symposium on light in horticultural systems. ISHS Acta Horticulturae 956. Nhut, Duong Tan, Nguyen Trinh Don, Nguyen Hong Vu, Nguyen Quoc Thien, Dang Thi Thu Thuy, Nguyen Duy, and Jaime A. Teixeira da Silva. 2006. Advanced technology in micropropagation of some important plants. Pages 325-335 in Jaime A. Teixeira da Silva ed. Floriculture, Ornamental and Plant Biotechnology Volume II. Global Science Books, UK. Osram Sylvania. 2000. Technical information bulletin: lights and plants, standard wide spectrum Sylvania Gro-Lux Flourescent Lamps. Available at: http://assets.sylvania.com/assets/documents/FAQ0074-0605.844b0c66-0b11-44c1-b6b5- 32218c3e6d08.pdf Perveen, S., A. Varshney, M. Anis, and I. M. Aref. 2011. Influence of cytokinins, basal media and pH on adventitious shoot regeneration from excised root cultures of Albizia lebbeck. Journal of Forestry Research 47-52. Petrus-Vancea, Adriana, and Dorina Cachita-Cosmo. 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) 18:81-86.
- 17. Griffith 17 Polking, Gary F., and Loren C. Stephens. 1995. Plant micropropagation using african violet leaves. Available at: http://www.biotech.iastate.edu/publications/lab_protocols/AV_Micropropagation.html Reed, Barbara M., and Piyarak Tanprasert. 1995. Detection and control of bacterial contaminants of plant tissue cultures. A review of recent literature. Plant Tissue Culture and Biotechnology 3:137-142. Rout, G. R., A. Mohapatra, and S. Mohan Jain. 2006. Tissue culture of ornamental pot plant: A critical review on present scenario and future prospects. Biotechnology Advances 24:531-560. Schubert, E. Fred., and Jong Kyu Kim. 2005. Solid-State Light Sources Getting Smart. Science 308:1274-1278. Seibert, Michael, Phyllis J. Wetherbee, and Donald D. Job. 1975. The effects of light intensity and spectral quality on growth and shoot initiation in tobacco callus. Plant Physiology 56:130-139. Skirvin, Robert M., Mel C. Chu, Mary L. Mann, Heather Young, Joseph Sullivan, and Thomas Fermanian. 1986. Stability of tissue culture medium pH as a function of autoclaving, time, and cultured plant material. Plant Cell Reports. 5:292-294. Wichada, Sunpui, and Kanchanapoom Kamnoon. 2002. Plant regeneration from petiole and leaf of African violet (Saintpaulia ionantha Wendl.) cultured in vitro. Songklanakarin Journal of Science and Technology 24(3):357-364.
- 18. Griffith 18 Taha, R. M., N. Duad, and N. A. Hasbullah. (Undated). Establishment of efficient regeneration system, acclimatization and somaclonal variation in Saintpaulia ionantha H. Wendl. Acta Horticulturae. Available at: http://actahort.org/books/865/865_14.htm Taiz, Lincoln, and Eduardo Zeiger. 2010. Working with light. Available at: http://5e.plantphys.net/article.php?ch=t&id=131