RESPONSE OF WALLISII REGEL. AND PHILODENDRON SCANDENS C. KOCH AND H. SELLO TO GAMMA IRRADIATION

By

MOHAMED ABD EL-SAMIAA SAYED KHALIFA B.Sc. Agric. Sci. (Ornamental Horticulture), Fac. Agric., Cairo Univ., 2010

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

In

Agricultural Sciences (Ornamental Horticulture)

Department of Ornamental Horticulture Faculty of Agriculture Cairo University EGYPT

2016

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INTRODUCTION

Peace lily ( Regel) is a member of the family and one of the most popular indoor (Sardoei, 2014). It originates from Panama, Columbia, Ecuador, Venezuela, the Malay Archipelago, Costa Rica, and the Philippines where it thrives in humid, tropical rainforest understories. Peace Lily is an herbaceous evergreen with short, erect to creeping stems bearing tufts of foliage. are dark green and ovate to lanceolate. Wide selection of are ranging in height from 12 inches to 4 feet From an ornamental viewpoint, the spathes and spadices are called rather than the tiny true flowers on the spadix. Inflorescences are produced seasonally or intermittently and can also be induced with chemical sprays. NASA even praised them in the clean air study for their ability to remove formaldehyde, benzene, and carbon monoxide from interior air (McConnell et al. ,2003). Interest in peace lily is steadily increasing as it is a shade tolerant indoor , dark green foliage and white spathes. The showy white spathes of Spathiphyllum enhance its popularity and marketing as a “flowering” foliage plant (Henny et al., 2004). Although it was initially a plant for container, in recent years, the culture of this plant has been greatly expanded for the production of cut flowers (Manda et al., 2014). Mature plants produce attractive white flowers that last several weeks. The natural flowering period for Spathiphyllum is generally Jan. through June. Natural production begins to decline over the summer and is at its lowest

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level during the fall months (Henny, 1998). Environmental conditions and cultural practices for commercial production of Spathiphyllum have been established over the last 10 years. Conover (1992) recommended that plants should be produced under a photosynthetic photon flux density of 200 to 400 µmol m-2 s-1 and a temperature range of 20 to 32ºC.

Philodendron is native to tropical regions of NorthSouth and Central America. The plant diversity is 250 that comprise the genus. Although the genus name means “tree lover” in latin, many Philodendron species are herbaceous tree climbing vines, the genus also includes woody-stemmed plants that may reach 15 feet in height. In addition to great differences in size, leaves of recently developed cultivars vary in color from dark red-black to light orange. (Chen et al., 2003). Heartleaf philodendron, Philodendron scandens, (Araceae family), is one of the most important ornamental foliage plants. It is an evergreen climber, with solid green leaves, a good and widely used for potted plants, totem poles, and hanging baskets. It thrives well in low light intensity, humid and temperate (Bailey and Bailey, 1976, Chen et al., 2005 and Gilman, 2011). Mature plants rarely flower; therefore, no reports of hybridization exist for this species (Henny et al., 2010). Cultural requirements for hear tleaf philodendron have been well documented previously (Conover and Poole, 1974). Heartleaf philodendron is normally propagated asexually by single eye cuttings. Vines do not develop secondary branches; growers

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stick multiple eyes per container to obtain a full appearance in the final product.

Genetic variation is necessary in any plant breeding program for crop improvement. Induced mutations are highly effective to enhance natural genetic resources and have successfully assisted in developing improved and new cultivars among both seed and vegetatively propagated crops. Mutations occur in cells in two ways. Firstly, by alteration in nuclear DNA which is also known as point mutation and it cause addition, deletion, transition and transversion in nucleolus of cell. Thus it changes the structure of nuclear DNA and mutations occurred. In second way, the mutagen makes the change in cytoplasmic DNA that is also called as cytoplasmic mutation. Gamma rays are used for improving growth and quality of plants, for their high mutation frequency that affect morphology, anatomy, biochemistry, and physiology of plants (Chahal and Gosal, 2002).

Mutation breeding by gamma rays is one of the most powerful methods for developing new varieties, which is very successful in ornamental crops. Mohan Jain (2006) stated that induced mutations are highly effective to enhance natural genetic resources for vegetatively propagated crops. Several improvements in ornamental crops could be achieved by gamma mutation, including characters of flower and leaves, growth habit and physiological traits (Schum and Prell, 1998). Radiation mediated morphological, structural and functional changes in a plant are

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governed by the intensity and duration of the gamma rays which generally induce cytological, biochemical, physiological, morphological and genetically changes in cells and tissues (Rahimi and Bahrani, 2011; Jan et al., 2012 ; Haris and Jusoff, 2013 and Chandrashekar et al., 2013 ). Ornamental plants are ideal for the application of mutations induction techniques because many economically important traits. Induced mutations in ornamentals comprise traits, such as altered flower characters (colour, size, morphology, fragrance); leaf characters (form, size, pigmentation); growth habit (compact, climbing, branching); and physiological traits such as changes in photoperiodic response, early flowering, free-flowering, flower keeping quality, and tolerance to biotic and abiotic stresses. They are easily monitored after the mutagenic treatment. Furthermore, many ornamental species are heterozygous and often propagated vegetatively; this allows the detection, selection and conservation of mutants in the M1 generation. Kurimato et al. (2010) found that the age at the time of radiation exposure plays an important role in integrating radiation effects. The gamma rays at high doses disturb the leaf gas exchange, hormone balance, water exchange and enzyme activities (Kiong et al, 2008). It has been suggested to use gamma rays at lower doses (1-10 Krad) to obtain useful mutants.

The aim of this work was to study the effect of gamma irradiation treatments on growth, pigments, anatomical structure and DNA polymorphism, as well to induce genetic variations in

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two important indoor house plants, viz., peace lily (Spathiphyllum wallisii Regel) and heartleaf philodendron (Philodendron scandens C. kock&H. Sello) plants and to apply RAPD analysis for genetic polymorphism among the different obtained variants.

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MATERIALS AND METHODS

This research was carried out in the greenhouse of the Ornamental Hort. Depart., Fac. Agric., Cairo Univ., Egypt, during the period from 2013 to 2015, to study the effect of gamma irradiation on growth, pigment contents and molecular aspects of Philodendron scandens and Spathiphyllum wallsii plants.

1- Effect of gamma irradiation on growth, pigment contents and leaf structure of (6-month-old) Spathiphyllum wallsii plants

2- Effect of gamma irradiation on growth and pigment contents of (3-month-old) Spathiphyllum wallsii plants

Plant material The plants of the two ages were obtained from Egypt-Green Farm, Giza, Egypt. Gamma ray treatments:

The plants of the two ages (6-month and 3-month old) were irradiated with 0, 0.5, 4 or 8 krad gamma rays, using a C137 source from a unit gamma chamber at a dose rate of 1 krad/13 sec. at the National Center for Radiation Res. and Tech., Nasr City, Cairo, Egypt.

The experiment: On 17 July, 2013, the irradiated plants of the two ages were planted individually in plastic pots (20cm-diameter)

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using a mixture of sand + peat+ perlite (1/2/1, v/v) and set under a high-humidity propagation unit (plastic tunnel) with intermittent mist for a month. After a month, on 16 August, the plants were moved to raised benches in the greenhouse. NPK fertilizer (Krestalon, 19-19-19) was applied (monthly) to pots at 1.5 g/pot. Irrigation was done as the plants needed.

Data recorded

Data were recorded at two dates for the two ages as follows: a. After 6 months of growth on the following characters:

1. Survival %. 2. Plant height. 3. Stem diameter. 4. Number of leaves/plant. 5. Leaf area. 6. Petiol length. b. After 12 months of growth on the following characters:

1. Survival %. 2. Plant height. 3. Stem diameter. 4. Number of leaves / plant. 5. Leaf area. 6. Petiol length. 7. Fresh weights of plant, leaves and stems.

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8. Leaves/stem ratios (fresh weight). 9. Pigment content. 10. Leaf anatomical structure. The second Experiment: Effect of gamma irradiation on growth, physiological and molecular aspects of Philodendron scandens plant Plant material Mother plants (stock) of Philodendron scandens (in 30cm plastic pots) were obtained from Egypt-Green Farm, Giza, Egypt. The stock plants were located in a greenhouse. On 17 July, 2013, stem cuttings (10-12 cm height) were obtained from the mother plants. Gamma ray treatments Stem cuttings were irradiated with 0, 0.5,2, 4 or 8 krad gamma rays, using a Co60 source from a unit gamma chamber at a dose rate of 1 krad/13 sec. at the National Center for Radiation Res. and Tech., Nasr City, Cairo, Egypt.

The experiment The irradiated cuttings were stuck in trays, using a mixture of sand + peat (1/2, v/v) and forced to root under a high-humidity propagation unit (plastic tunnel) with intermittent mist. After a month, on August, the rooted cuttings were individually transplanted into 20-cm plastic pots, using a mixture of sand+ peatmoss+ perlite (1/2/, v/v) and set on raised benches in the greenhouse. NPK fertilizer (Krestalon, 19-19-19) was surface applied (monthly) to pots at 1.5 g/pot.

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Statistical analysis A randomized complete block design with 3 replications was used. Each treatment consisted of ninty of treated plants. Data were statistically analyzed for significance by analysis of variance (ANOVA) and means were compared using LSD at 5% level of significance, as described by Steel and Torri (1980).

Data recorded: After 12 months of growth the following characters were recorded:

1. Survival %. 2. Plant height. 3. Stem diameter. 4. Number of leaves /plant. 5. Leaf area. 6. Petiole length. 7. Fresh weights of plant, leaves and stems. 8. Leaves/stem ratios (fresh weight). 9. Pigment content. 10. RAPD-PCR- analysis of variants.

11. Rooting of cuttings of Philodendron scandens (M1V2).

After 12 months of growth, the rootability of cuttings for

treated and untreated plants was studied (M1V2). The cuttings were prepared at the length of 12-14 cm, with 3 nodes and 2 leaves.Then, the cuttings were planted in plastic pots filled with a mixture of sand + peatmoss (1/2, v/v) and

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forced to root under a high-humidity propagation unit (plastic tunnel) with intermittent mist.

Data recorded: After 60 days from planting the cuttings, the following characters were recorde; Plant height, survival %, number of leaves/ plant, stem diameter, number of roots /cutting and root length. Determination of pigments Chlorophyll a, b, and carotenoids were determined by the spectrophotometric method according to Saric et al. (1967) and Lichtenthaler (1987). The irradiated and non-irradiated plantlets were added to a pre-chilled mortar in an ice bath. The plantlets were ground with pestle after adding calcium carbonate (Spectrum, CA) at a ratio of 3 1g of plantlets to 2 g of calcium carbonate together with 10 ml of 80% (v/v) acetone. The sample extracts were filtered using Whatman no. 1 filter paper and followed by washing with 80% (v/v) acetone. The extraction volume was made up to 50ml with 80% (v/v) acetone. Sample extracts were subjected to spectrophotometric determination (Bio- Rad smart spec plus, USA) of chlorophyll at 646 nm and 663 nm.

The chlorophyll a and b chlorophyll b content in milligram per liter was determined according to the formula below and further expressed in milligram per gram fresh weight of plant material. - Chlorophyll a = 12.25(663nm) – 2.79(646 nm) - Chlorophyll b = 21.50(646 nm) – 5.10(663 nm)

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- Total chlorophyll, a + b = 7.15(663 nm) + 18.71(646 nm)

- Carotenoids = 4.695(440nm)-0.268(a+b)

Leaf anatomical structure of Spathiphyllum wallsii plants

For the anatomical studies of Spathiphyllum wallsii leaves (blade, petiole and stipule), samples of gamma treated and untreated leaves were fixed and killed for at least 48 hours in F.A.A. (10 ml formalin, 5 ml glacial acetic acid, 50 ml ethyl alcohol 95%, and 35 ml distilled water), washed in 50% ethenol, dehydrated in normal Butyl alchohol series (Sass, 1951) and embedded in baraffin wax. Sections were cut to a thickness of 20 microns and stained with safranin /light green combination and mounted in Canada balsam, according to Nassar and El-Sahar, (1998). The slides were microscopically examined. Counts and measurements (μm) of the different tissues (leaves, stems and roots) were taken and calculated using a micrometer eye piece. The surfaces of Spathyphyllum wallsii were pictured using light microscope with camera model Leica ICC50 HD at Faculty of Agriculture Research Park, Cairo University.

Molecular analysis (RAPD-PCR): RAPD-PCR analysis for Philodendron scandens variants was done as follows: CTAB - DNA Extraction Protocol (Doyle & Doyle, 1987) Before Beginning:

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1. Prepare CTAB Buffer (recipe attached). Prior to starting extraction, add polyvinylpyrrolidone and -mercaptoethanol. CTAB Buffer PVP -mercaptoethanol 0.5 ml 0.02 g 2.5 µl 2. Grind 50-60 mg tissue in liquid nitrogen with blue pestles, keeping tissue frozen the entire time. 3. Add 500 uL of CTAB buffer and mix the tubes. Incubate at 55°C for 3 hours, mixing once after 30 minutes. 4. After incubating, add 1.5 µl RNase A. Incubate at 37°C for 15 minutes. 5. Remove samples from water bath and add 500 µl of chloroform and mix by gently shaking tubes. 6. Centrifuge for 7 minutes at 16 rcf. 7. Transfer the aqueous phase into the new tube. 8. Add 0.1 volumes cold 7.5 M ammonium acetate. 9. Add 0.6 volumes of cold isopropanol. Mix by inverting tubes 20-30 times. 10. Incubate on ice for 60 minutes. 11. Centrifuge for 3 minutes at 16 rcf. 12. Discard supernatant and add 700 µl 70% EtOH, invert tubes 5-10 times. 13. Centrifuge for 1 minute at 16 rcf. 14. Discard supernatant. 15. Hydrate pellets with 50 µl TE.

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1. Isolation of genomic DNA: DNA isolation was performed from treated and untreated leaves using CTAB method (Doyle and Doyle, 1987).Two grams of fresh leaves were ground to fine powder in liquid nitrogen, the powder was then placed in tubes containing 5 ml pre-heated CTAB extraction buffer, incubated at 65°C for 45 min., gently shacked every 15 min. DNA was extracted in chloroform-iso amyl alcohol (24:1v/v),then it was centrifuged for 10 min. (12,000 rpm),and 600 μl of the supernatant were transferred to tube, following the addition of 500 μl chloroform-iso amyl alcohol(24:1v/v). Table (a): Primer names and their nucleotide sequences assayed in RAPD-PCR. No. Primer Sequence (5΄-3΄) 1 A01 CCCAAGGTCC 2 B10 CAGGCACTAG 3 S18 CCACAGCAGT 4 S20 GGACCCTTAC 5 N8 ACCTCAGCTC 6 H5 AGTCGTCCCC

2. RAPD-PCR reaction It was performed, stained, photographed according to Roehrdans and Flanders (1993), 6 primers were used in this study (Table, a). Reaction and conditions of RAPD-PCR were the following: 1. Gently vortex and briefly centrifuge all solutions after thawing.

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2. Place a thin-walled PCR tube on ice and add the following components for each 25 µl reaction:

10X DreamTaq Buffer* 2.5 µl dNTP Mix, 2 mM each (#R0241) 2 µl

Primer 2 µl

25 mm MgCl2 (#R0971) 3 µl

DreamTaq DNA Polymerase 0.25 µl

Template DNA 1 µl

Water, nuclease-free (#R0581) 14.25 µl

Total volume 5 25 µL

3. Gently vortex the samples and spin down.

5. Place the reactions in a thermal cycler. Perform PCR using the recommended thermal cycling conditions outlined below:

Temperature Number of Step Time (°C) cycles

Initial denaturation 92 5 min 1

Denaturation 92 30 sec

Annealing 35 1 min 40

ramp up to 72º C 5 min

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Extension 72 2 min

Final extension 72 10 min 1

3. Electrophoresis: The amplification products were analyzed according to Sambrook et al. (1989) in 2 % agarose in TAE buffer, nucleic acids bands were photographed and detected

under UV light.

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RESULTES

The present study was carried during the period from 2013 to 2015, at Ornamental Horticulture Department, Faculty of Agriculture, Cairo University, to study the effect of gamma radiation on growth characteristics, morphological variations, pigments and molecular aspects of Spathyphyllum wallisii and Philodendron scandens plants. 1- Effect of gamma radiation on Spathyphyllum wallisii plants The aim of this study was to investigate the effect of gamma irradiation on growth, pigment, abnormalities and leaf structure of Spathyphyllum wallsii plants at two ages (6-month-old and 3-month- old, plants). The plants of the two ages were irradiated with 0, 0.5, 2, 4 or 8 krad gamma rays, using a Cesium 137 source. This experiment was divided into two parts. The irradiated plants were planted individually in plastic pots (20cm-diameter) using a mixture of sand + peat+ perlite (1/2/1, v/v) in the greenhouse, the data were recorded after 6 and 12 months from gamma radiation treatments. Plant and leaves abnormalities and anatomical structure of the leaf, petiole and stipule were recorded. The results of this study can be summarized as follows: 1. Effect of gamma radiation on 6-month-old of Spathiphyllum wallisii plants After 6 months, exposure plants to gamma radiation at 4 or 8 Krad, significantly reduced the survival % compared with control, whereas, after 12 months, all gamma doses caused a significantly reduced it. Plant height and stem diameter, after 6 and 12 months,

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were significantly decreased with all gamma doses treatments. After 6 months the 0.5 and, 4 Krad doses of gamma rays insignificantly decreased the formation of suckers, but after 12 months all the treatments reduced it. Leaf length and leaf width, after 6 and 12 months, were significantly decreased with all gamma doses treatments.The low dose of gamma radiation (0.5 Krad) gave significantly the shortest petiole after 6 months from treatments, after 12 months all the gamma rays treatments significantly reduced it. Number of leaves was significantly decreased at the dose of 0.5Krad, as compared with control but the doses of 4 Krad and 8 Krad significantly increased it. Leaf area decreased significantly with the different doses of gamma rays, the smallest leaf area recorded with 8 Krad treatment. Treating plants with 0.5 and 4 Krad gamma radiation significantly reduced the fresh weight of leaves and the dose of 4 Krad gamma radiation significantly reduced the dry weight of leaves. All gamma doses increased the stem length and had insignificant effect on stem thickness, fresh and dry weights of stem, as compared with the control. Number of roots decreased with the different gamma doses, as compared with control. Whereas, using gamma at doses of 0.5 or 4 Krad, insignificantly decreased the root length. All gamma doses decreased the fresh weight of roots and had no significant effect dry weight, as compared with control. All gamma treatments insignificantly increased the content of chlorophyll (a), chlorophyll-

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b and total chlorophylls, and the 4 or 8 Krad gamma doses significantly increased the content of carotenoids, compared with control. The gamma rays treatment at the dose of 0.5 Krad increased he thickness of leaf blade (lamina) to maximum value and increased the stomata size as compared with the other gamma radiation treatments and the control. All gamma radiation treatment the thickness of petiole.The thickest petiole was recorded with the dose of 4 Krad. The doses of 0.5 and 4 Krad increased the number of vascular bundles of leaf petiole, compared with the control .The highest number of vascular bundles was recorded by treating the plants with gamma rays at 0.5 Krad. The treatment of gamma radiation at 4 Krad gave the thickest midrib followed by the gamma treatment of 0.5 Krad. Whereas, the dose 8 krad, gave the smallest diameter, compared with the control. The stipule length and the number of vascular bundles increased with the gamma treatments at dose of 0.5 Krad, in comparison with the control. 2. Effect of gamma radiation on 3–month-old of Spathiphyllum wallisii plants. After 6 months exposure plants to gamma rays at 0.5 Krad insignificantly reduced the survival percentage, compared with control, after 12 months, all the gamma treatments significantly reduced it. After 6 and 12 months from the gamma treatments, the doses of 4 and 8 Krad significantly increased plant height, compared with the control. Suckers number /plant reduced after 6 months the different doses of gamma significantly decreased the

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formation of suckers, but after 12 months this effect was insignificant, compared with the control. Treating the plants with gamma at the doses of 4 or 8 Krad increased the leaf length and leaf width .The dose of 8 Krad, gave the longest leaves,the dose of 4 Krad gave the highest value of leaf width and the dose of 0.5 Krad gave the largest leaf area. The treatment of 0.5 Krad gamma gave the shortest petiole, whereas, treating the plants with 4 or 8 Krad of the gamma increased it. Treating plants with gamma rays at the dose of 4 Krad significantly increased the formation of leaves/plant, as compared with control. The different doses of gamma increased the fresh weight of leaves, the dose of 4 Krad gave the heaviest leaves. Treating plants with the doses of 0.5 or 4 Krad insignificantly increased the dry weight of leaves, in comparison with the control. All gamma doses had no significant effect on stem length and the low gamma dose 0.5 Krad significantly increased the stem thickness, whereas the doses of .4 Krad to 8 Krad, significantly reduced it, as compared with the control. Fresh weight of stem increased with the gamma at doses 0.5 or 4 Krad, the lowest fresh weight of stem recorded with the dose of 8 Krad.The heaviest dry weight of stem recorded with the dose of 0.5 Krad. All gamma rays treatments had no significant effect on the number of roots/plant, fresh and dry weights of roots, but insignificantly decreased the root length, as compared with the control .The dose of 8 Krad gave the longest roots. All gamma doses insignificantly decreased the content of chlorophyll (a),

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chlorophyll (b) and total chlorophylls as well as the content of carotenoids. 3. Effect of gamma radiation on Philodendron scandens plants. The aim of this study was to investigate the effect of gamma irradiation on growth, pigment, abnormalities and genetic variation (PCR-analysis) of Philodendron scandens plants. Stem cuttings (10- 12 cm length) of the plants were subjected to four doses of gamma irradiation (0.5, 2, 4 or 8 Krad) and 0 as a control, using a cobalt 60 source The irradiated cuttings were stuck in trays, using a mixture of sand + peat (1/2, v/v) and forced to root under a high-humidity propagation unit (plastic tunnel) with intermittent mist. After a month, the rooted cuttings were individually transplanted into 20-cm plastic pots, using a mixture of sand + peatmoss + perlite (1/2/1, v/v). The results of this experiment can be summarized as follows : All doses of the gamma radiation decreased the survival %, which gradually decreased as the gamma dosage increased. The plant height, number of leaves/ plant, and fresh weight of plants, leaves and stems were decreased with the different doses of gamma rays. The highest average leaf weight and leaf area were obtained by the low dose of the gamma radiation (0.5 Krad), while higher doses decreased it. The highest dose of the gamma radiation (8 Krad) caused some morphological variations. Biochemical studies revealed that the highest contents of chlorophylls (a and b) and carotenoids were determined from application of higher doses of the gamma radiation , as compared

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with the control. The pplication of 0.5 Krad of the gamma radiation treatments decreased the content of carotenoids to the lowest value and the highest value was determined with the gamma radiation treatment of 8 Krad. Changes in DNA caused by gamma irradiation resulting in genetic variations were detected by RAPD-PCR profiles, performed using 6 arbitrary primers, which detected polymorphisms between the control and the plants treated with gamma rays at different doses. It is worth to mention that, the dose of gamma rays at 0.5 Krad gave the highest number of DNA fragments and showed desirable genetic effects on some morphological traits. Leaf abnormality of Philodendron plant in response to gamma radiation treatments at the doses 0.5, 2, 4 and 8 Krad were irregular leaf thickness, asymmetric development of blade, reduction in number of leaves, chlorophyll variegation, narrow leaves and small leaves. Unequal development of lamina. The number of abnormal leaves and plants increased with the low doses of gamma rays (i.e.0.5 and 2 Krad) and asymmetric development of blade was the common abnormality observed.

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