INTRODUCTION

Catharanthus roseus (L.) G. Don. (Madagascar periwinkle)  is a tropical and subtropical ornamental plant  and  one of the most important medicinal plants (also known as anticancerous drug yield  in plant)  and also  an ornamental bedding plant belonging to the family Apocyanaceae (Jaleel et al.,2008).

Catharanthus roseus contains a virtual cornucopia of useful alkaloids, used in diabetes, blood pressure, asthma, constipation, and cancer and menetrual problems. There are about two common cultivars of C. roseus which is named on the basis of their flower colour that is the pink flowered “Rosea” and the white flowers “Alba”. Catharanthus roseus is found to be a species of Catharanthus native and also endemic to Madagascar. The synonyms of the plant name include Vinca rosea, Ammocallis  rosea and Lochnera  rosea, other English names occasionally used for the plant include Cape  periwinkle, rose periwinkle, rosy periwinkle and “old maid”.

Catharanthus roseus is an evergreen subherb or herbaceous plant growing to 1 m. tall. The leaves are oval to oblong, 2.5- 9.0 cm long and 1- 3.5 cm broad, glossy green hairless with a pale midrib and a short petiole about 1- 1.8 cm long arranged in opposite pairs. The flowers are white to dark pink with a dark red center; with a basal tube about 2.5- 3 cm long and a corolla about 2-5 cm diameter with five petal like lobes. The fruit is a pair of follicles about 2-4 cm  long and 3 mm broad.

Catharanthus  roseusposses carbohydrates, flavinoids, saponins and alkaloids. Alkaloids are the most potentially active chemical constituents of Catharanthus  roseus. More than 400 alkaloids are present in the plant, which are used as pharmaceuticals, agrochemicals, flavor and fragrance ingredients, food additives and pesticides. The alkaloids like actineoplastidemeric, Vinblastin, Vincrestine, Vindesine, Vindeline Tabersonine etc. are mainly present in aerial parts whereas ajmalicine, vinceine, vineamine, raubasin, reserpine, catharanthine  etc are present in roots and basal stem. Rosindin is an anthocyanin pigment found in the flower of C. roseus(Sain and Sharma, 2013).

Aim of the work:

1. Studying the effect of different doses of gamma radiation from cobalt 60  and salt water treatments on  the vegetative and flowering growth of  Catharanthus roseus , as well as on the possibility of   inducing  mutations , which  can resist high salinity or  have wider  landscape  value .
2. Selecting a new strain of Catharanthus roseus, with high alkaloid productivity.
3. Using of isozymes techniques (peroxidase enzymes) to find out the genetic relationship among the original mother plant and the mutated plants.

MATERIALS AND METHODS

The experiments were carried out in the Flowers and Ornamental Plants Research Gardens, Department of the Floriculture and Ornamental Horticulture and Landscape Gardening, Faculty of Agriculture, Alexandria University during 2013 – 2015.

Materials

Plant materials

Local cultivar of Madagascar periwinkle or rosy periwinkle (Catharanthus  roseus (L.) G.Don was used in this study, with purple flowers and cross-pollination.  Seeds were obtained from the Flowers and Ornamental Plants Research Gardens of the Faculty of Agriculture, University of Alexandria.

Gamma radiation source

Gamma - rays doses applied in this study were generated from the Cobalt 60 Source, in Gamma – Cell installed in the Irradiation Laboratory at Middle East Regional Radio-Isotope Center for Arab Countries, El-Dokky, and Cairo, Egypt.

Methods

a. Experimental design

The effects of the two factors ( irradiation and salinity ) on the M₁ - plants were tested in  field. The layout of  the experiment was designed as factorial layout in Randomized Complete Block Design (RCBD) (Gomez and Gomez, 1984) which contained 5 radiation treatments , i.e. control (0) , 5, 10, 15 and 20 kr.from  gamma rays   and 3 salinity levels of the irrigation water (0,100 and 150 mM NaCl).  One hundred and fifty seeds of Catharanthus  roseus were used for every  treatment from Gamma rays, One hundred and fifty seeds for every  salinity treatment within each gamma rays treatment.

b. Preparing of seeds

Lot of well developed pure seeds  from healthy and abundantly fruitful plants of  Madagascar periwinkle or rosy periwinkle (Catharanthus  roseus L.) Local cultivar were collected. The total amount of seeds prepared for gamma ray treatments  was divided into five equal portions; the first portion for control, while the  other four portions of seeds were, paged equally in four paper bags before exposure to radiation.

c. Gamma radiation practices

On the 18 ^th and 26 ^th of March 2013 and 2014 in the first and second seasons; respectively, the dry seeds of Catharanthus  roseus L.were exposed to four different doses of gamma rays as 5, 10, 15 and 20 kr from Co-60.

d. Soil analyses

Physical and chemical analyses of the used soil were carried out according to the standard methods outlined by Page et al. (1982) and are listed in Table (1).

Table (1). Some Physical and chemical characteristics of the used soil during 2013 and 2014.

The experimental treatments consisted of two salinity levels of the irrigation water (100 and 150 mM NaCl) in addition to control (0.0 mM), salinity levels were obtained by addition of appropriate amount of dry NaCl to water. The salinity levels were equivalent to an electrical conductivity of 0.46, 10.9 and 15.9 dSm^-1, respectively using a portable EC meter instrument. To avoid an osmotic shock for seedling emergence; the salinized water was used after 45 days of sowing (Gorham and Wyn Jones, 1993).

To prepare the stock  solution, a commercial sea salt (sodium chloride) without  purification (contents: NaCl  98.5% , Humidity 0.3% and  KIO₃ 30-70 ppm) produced by  Egyptian salt and mineral company (EMISAL) was dissolved in tap water (0.46 dS/m) at (5.85 g salt per liter =100 mM and 8.775 g salt per liter =150 mM).One month later, complete fertilizer 19-19-19 was top dressed at the rate of 1/2 g /l and this addition was repeated every two weeks.

The plants were irrigated 3 times weekly in summer with 320 ml  per pot until the end of the experiment.

Cultural aspects

a. M₁ - Generation

Gamma- rays treated and non - treated  seeds were sown  on March 20,  2013  in  the first season and on March 27,2014 in  the second one. The seeds of each treatment were sown in three trays (150 seeds) filled with a mixture of equal parts of sand  and clay(1/1).The trays were placed in partial shade according to the factorial experimental  layout  of the Randomized Complete Block Design and watered daily. On May 3 , 2013 and April  22, 2014 in both seasons , the trays were gradually transferred from shade to open place (sunny place)  for one week on May 10, 2013 and  April 29, 2014 in the first and second seasons, respectively. Two seedlings were transplanted into 30 cm diameter plastic pot containing   sandy loam soil and reached a height of about ten cm. The pots were arranged in the three replicates according to the Randomized Complete Block Design with different numbers of pots in each treatment according to the number of the survived seedlings. 

b. M₂ - Generation

For growing the M₂- generation in both seasons ,  seeds were  collected from  each  treatment on  March 20, 2014 and March 23, 2015 in the first and second seasons, respectively, and sown in three trays( 100 seeds for each treatment). The trays contained a soil mixture of   1 sand: 1 clay by volume. The trays were placed in partial shade according to the factorial experimental layout of Randomized Completely Block Design with 3 replicates (Gomez and Gomez, 1984).The trays were watered daily.  On April 24 , 2014 and April 28, 2015 in the first and second seasons ; respectively , the trays were gradually transferred from shade to sunny place along one week . On April 24, 2014 and April 8, 2015 during the first and second seasons; respectively, every two M₂- seedlings were transplanted into a plastic pot of 30 cm diameter, containing   sandy loam soil and reached a height of about ten cm.  The pots were arranged according to the experimental design mentioned before.

Experimental Data

The following parameters were recorded in both M₁ - and M₂-generations of the two successive experimental seasons.

1. Morphological characteristics, such as habit of growth, leaf size, form and colour, stem colour and flower structure and colour.
2. Leaf proline content (according to Bates et al. 1973).
3. Leaf  alkaloids,  vindolen and  catharanthine contents  ( after Luo et al., 2005)
4. Leaf  total carbohydrates content

     Total leaf carbohydrates content was determined colorimetricaly as reported by Loomis and Shull (1937) and Dubios et al.,(l956).

1. Variations and Mutations.

 All plants of the different treatments in both M₁ and M₂ experiments were examined daily to search for the variation. Changes  in the vegetative or flowering growth were recorded. These changes included :

a)  Habit of growth.

b)  Leaf colour and form.

c)  Flower colour and form.

6 .Peroxidase isozyme electrophoresis

The gamma rays and salinity treatments caused variation in the flowers form and tolerance   to salinity compared with the control. Leaves were used for the isozymes techniques from the control and the mutated twelve plants. The peroxidase isozymes patterns were examined after the method described by Sabrah and El- Metainy (1985).

RESULTS AND DISCUSSION

1. Effect of gamma radiation and salinity on the morphological characteristics

Some  variations in seed germination percentage ,plant height, internode length, stem diameter , number of branches, number of leaves, leaf area, specific leaf weight, total leaf chlorophyll content  (a,b and a+b), total carotene, fresh and dry weights of the plants, flowering date ,number of flowers per plant, flowering  period, flower length and diameter, pollen viability, survival, fresh and dry weights of the roots, were recorded as a result of different  treatments

1. Effect of the gamma  radiation  and salinity on  the  leaf proline content

The analysis of variance  showed that the effects of the gamma radiation, salinity   treatments  and  interaction  between them  were  highly  significant  on the leaf  proline content  in the M₁ and M_{2 -}generations of  the second season.

Data on the effect of gamma radiation  and salinity treatments on  the leaf proline content   of the M₁ and M_{2 -} generations of  the second season are listed in Table  2.

In the M_{1 -}generation there were highly significant differences among gamma rays treatments. The highest average was at the 20 kr treatment (0.4052 g/100g) and the lowest one was at 0 kr (0.2296 g/100g).By the salinity treatments, there were also highly significant differences. The highest average was at the 150 mM (0.4497 g/100g) and the lowest one was at 100 mM (0.2376 g/100g). The interaction was   also highly significant. The 20 kr with 100 mM had the highest proline content (0.7690 g/100g). The lowest one was at the treatment of 20 kr with 0 mM (0.0006 g/100g) (Table 2).

In the M₂ – generation, there were highly significant difference among the gamma rays treatments. The highest average by  the  gamma rays  was at the 20 kr treatment (0.9993 g/100g)  and  the lowest average was  at 5 kr (0.3579 g/100g).The salinity treatments  caused  highly significant differences. The highest average was at the 150 mM (1.0788 g/100g) and the lowest average was at 0 mM (control) treatment (0.3789 g/100g). The interaction was also highly significant. The 20 kr with 150 mM treatment had the highest proline content(1.4700 g/100g). The lowest average was at the treatment of 5 kr with 100 mM (0.1818 g/100g).

The results of gamma rays were similar to those reported by Desai and Rao (2014) on  Cajanus cajan . The results of the salinity treatments were similar to those reported by Zidan and Alzahrani (1994) on Ocimum basilicum L. and Heidari  and Sarani (2012)  on  Matricaria chamomilla.

Generally, the treatments  of  gamma rays  and salinity caused some increases in proline content in the M₁ and M₂ generation,  which is harmony with the results of  Nikam et al.(2015) on Saccharum officinarum L. which  can be used for the production of mutants  which have  the ability for environmental stress tolerance, Desai  and  Rao(2014).

The  results of this work revealed that  the dose  of 20 kr significantly   increased the amount of leaf proline  in the irradiated plants  comparing  with  the control in  the  M₁- and M₂ – generations  of the second seasaon. Higher  level  of proline content   in leaves may  be due to irradition  at  20 kr stimulated  the experession of genes encoding  enzymes of proline synthesis  such  as  pyrroline -5- carboxylate.  Also, irradiation decreased enzymes of proline    oxidative such as proline dehydrogenase. This explanation is similar to the opinion mentioned by Amini and Ehasapour (2005).

The  results of the  M₂  -generation during   the second  season  declared that the  doses  of 5,10 and 15 kr significantly reduced  the amount of leaf  proline  in  irradiated  plants  as compared to  the  control.This  reduction in leaf  proline  content could be  attributed to the  inhibition  effect  of gamma-rays  doses    mentioned  before on  the  expression  of genes encoding enzymes of proline synthesis  and /or  enhancing the activity of enzymes of  proline   oxidative. This declaration is nearly   similar to that reported by Amini and Ehasapour (2005).

Table  (2). Average values of the leaf proline content of  Catharanthus    roseus, L.  as affected by gamma radiation   (kr) and  salinity levels  (mM) treatments in the M₁ – and M₂  generations of the second season.¹⁾.

¹⁾Values marked with the same alphabetical letters, within comparable group of  means, do not differ   significantly, using

L.S.D. at 0.05 level of probability.

It is  clear that the  salinity  treatments of  100 and 150mM significantly   increased  the  amount of  leaf proline  as compared  with  the control. It  has  been  established that the plants accumulate   a variety   of  osmoregulator   soluts  including proline as an  adaptive mechanism  to environmental stress and salinity (Aspinall  and Paley,1981). The use of  proline as  osmoregulator  to  overcame the  bad effects of salinity, which  is  similar  to the  effect of seawater on plant growth has been  reported  by  Lin and Kao(1996). Increase in proline content with  increasing  stress   is one of the defense mechanisms   which  is  used  by stressed   plants to  reduce cell osmotic  potential which  resulted  in increasing  cell water  uptake  with concomitant increases  in  cell  turgidity and  activity (Khalil and El- Noemani,2012). Stressed plants diminish osmotic potential by accumulating free amino acids,ions,proline,soluble protein and  carbohydtaye (Salama et al.,1994). These osmolytesmight increase the osmotic pressure of cytoplasm and enhance water flow into the different plant organs and tissues.

3. Effect of radiation   and salinity on  the leaf alkaloids

3a.Effect of gamma   radiation  and salinity on  the leaf vindolen content

The analysis of variance showed that the effect of the gamma radiation alone and the interaction between gamma radiation and salinity were not significant, but the effect of salinity treatments on the leaf vindolen content was significant in the M₁-generation.

Data on the effects of gamma radiation and salinity treatments on the   leaf vindolen content of the M₁ and M₂ –generations in second season are listed in Table 3a.

In the M₂-generation, the effects of the gamma radiation and that of theinteraction between gamma radiation and salinity    were highly significant but   the effect of the salinity treatments was only significant.

Table 3a presents the mean values of the leaf vindolen content of the different treatments. In the M₁-generation, there were no significant differences between gamma rays treatments. The highest average between the  gamma rays  was at the 20 kr treatment (1.37 mg/g) and  the lowest one was  at 0 kr (control) (1.27 mg/g).The effect of the  salinity treatments was significant. The highest average between the   treatments salinity was   at the control treatment (1.66 mg/g) and the lowest one was at 100 mM (0.97 mg/g). The 20kr with 0 mM treatment had the highest leaf vindolen content (2.12 mg/g). The lowest averages were at the treatment of 20 kr with 100 mM (0.86 mg/g).

In the M₂- generation, there were highly significant differences among the gamma rays treatments. The highest average was at the 20 kr treatment (3.35 mg/g) and the lowest one was at 10 kr (2.29 mg/g).The effects of the salinity treatments were significant. The highest average was at the 150 mM (2.94 mg/g) and the lowest one was at 0 mM (control) treatment (2.41 mg/g). The 20 kr with 150 mM treatment had the highest leaf vindolen content (4.20 mg/g). The lowest average was at the treatment of 15 kr with 0 mM (1.65 mg/g) (Table 3a).

The results of gamma rays were similar to those reported by Abdel-Hady et al.(2008)  on Atropa belladonna and Shaimaa et al.(2013)on  Brassica rapa at gamma rays.

Generally, the treatments of salinity caused some increases in leaf vindolen content in the M₂- generation,  which in harmony with the results of  Ali (1991) on Daturaand Heidari  and Sarani (2012) on  Matricaria  chamomilla.

Also  the effect of gamma -rays and salinity on the  vindolen   was  similar to  the result  reported by Shaimaa et al.(2013)on Brassica rapa.

Accumulation of alkaloids was considered as an adaptation to the imposed salinity stress because they have an osmoregulatory role (Elhaak and Wegmann, 1997).

William et al (1998) reported thatthe increase in the alkaloids content as the influence of NaCl is a combination of an osmotic effect and a specific ion effect. He added that the increase of alkaloids in response to salinity may be due to its role in the plant protection against the salt stress effects.

           Table (3a). Average values of the vindolen content of Catharanthus    roseus, L.  as affected by gamma radiation (kr) and  salinity levels (mM) treatments   in the M₁ – M₂  generation  of  second  season.¹⁾.

¹⁾Values marked with the same alphabetical letters, within comparable group of  means, do not differ   significantly, using

 L.S.D. at 0.05 level of  probability.

3b.Effect of the gamma radiation and salinity on the leaf catharanthine content

The analysis of variance   showed that the effects of the gamma radiation, salinity treatments and the interaction between them   on the leaf catharnthine content were highly significant in the M₁-generation in the second season.

Data on the effect of gamma radiation and salinity treatments on the leaf catharnthine content of the M₁ and M₂-generations   of the second season are listed in Table (3b).

In the M₂-generations ,the effect of the gamma radiation was not  significant,but that of the  salinity  treatment was significant,  while  the   interaction  between  them was highly  significant.

Table 4   presents the mean values of the leaf catharnthine content   of the different treatments. In the M₁-generation, there were highly significant differences among the gamma rays treatments. The highest average was at the 10kr treatment (0.413 mg/g) and the lowest one was at 15 kr (0.104 mg/g).The effects of the salinity treatments were highly significant. The highest average was  at the control treatment (0.372 mg/g) and  the lowest one was  at 100 mM (0.079 mg/g). The 10 kr with 150 mM had the highest leaf catharnthine content (0.890 mg/g). The lowest average was at the treatment of 20 kr with 100 mM (0.002 mg/g).

In the M₂- generation, there were no significant differences among the gamma rays treatments. The highest average between the gamma rays was at the 15 kr treatment (0.186 mg/g) and the lowest one was at 10 kr (0.116 mg/g).The effects of the salinity treatments were significant. The highest average was at the 100 mM (0.177 mg/g) and the lowest one was at 0 mM (control) treatment (0.128 mg/g). The 0 kr with 100 mM treatment had the highest leaf catharnthine content (0.316 mg/g), while the lowest averages  was at the treatment  of  10 kr with 150 mM (0.080 mg/g).

The results of this work indicated that the gamma doses of 5 and 10kr significantly increased the leaf catharnathine content compared with the control during the M₁-generation of the second season. It was also noticed that the dose of 20kr significantly increased the leaf vindolen content as compared   with the control during the M₂-generation of the second season. This means that radiation supported accumulation of alkaloids in the irradiated plants. The respone of plants against radiation  induced reproductive and metabolic disorder may  be  due to  the accumulation of several bioactive constituents like  alkaloids (Padhya,1986),which may act through different  mechanisms such as inhibition  of lipid peroxidation (Goel et al. 2004).

Alkaloids are end products for the reaction of toxic components in plants and they are harmless for plants (Hossien, 1987). The radiation may stimulate thesereaction which resulted in accumulation of alkaloids in the irradiated plants.

Regarding the salinity treatments, it was noticed that the treatment of 10mM in the M₂-generation   of the second season significantly reduced the amounts of leaf catharnathine and vindolen compared with the control. It  is  known that under stress  condiation  plants generally  shift a major  protion  of their metabolic  activies towards secondary  metabolite synthesis, so an increase  in alkaloid contents was  expected (Ali,1991;Moons et al. 1997;Wu et al.,2004;Pandey et al., 2007 and Shaimaa et al. ,2013).

But  in the case of the treatment of 100mM during  the M₁-generation  the decrease in alkaloids was  recorded and it was unexpected.During  the M₂-generation   of the  second season,it was  clear  that the treatment of 10mM significantly  increased the amount of the catharnathine and both treatments of 100 and  150mM significantly increased the amount  of leaf vindolen  as compared with the control. It  has been mentioned before  that under stress condition as salinity stress  plants generally  shift a mojor  protion of their metabolic activeties  towards  secondary metabolite  synthesis, as alkaloids (Ali,1991;Moons  et al., 1997;Wu et al.,2004;Pandey et al., 2007 and Shaimaa et al. ,2013). A biotic stresses as salinity stress may result in an increase in the level of endogenous methyl jasmonate, which can stimulate the activity of enzymes involved in the biosynthesis of alkaloids leading to enhanced alkaloids accumulation (Moons   et al., 1997).

   Table (3b). Average values of the leaf catharnthine content of Catharanthus roseus, L. as affected by gamma radiation (kr) and salinity levels (mM) treatments in the M₁ – M₂ generation of second season.¹⁾.

¹⁾ Values marked with the same alphabetical letters, within comparable group of  means, do not differ   significantly, using

 L.S.D. at 0.05   level of  probability

4. Effect of the gamma radiation and salinity on the total carbohydrate content

The analysis of variance showed  that the effects of the gamma radiation, salinity and  the interaction  between them  on the total carbohydrate   content was not significant in the M₁-generation in  the second season. In the M₂, the effects of the gamma radiation and salinity were significant but the interaction between them was not significant. Table 4 presents the mean values of the total carbohydrate   content of the different treatments in M₁-and M₂ of the second season. In the M₁-generation, there were no significant differences among the gamma rays treatments. The highest average was at the 0 kr (control) treatment (5.39 %) and the lowest one was at 20 kr (4.68 %).The effects of the salinity treatments were also not significant. The highest average between the  salinity  was  at 150 mM (5.38 %) and  the lowest one was  at 0 mM (control) treatment  (4.90 %) and  the interaction  between radiation  and  salinity  was  not significant. The 10 kr with 150 mM had the highest total carbohydrate content (6.72 %) and the lowest average was at the treatment of 10 kr with 0 mM (4.04 %) .In the M₂- generation, there was significant difference among the gamma rays treatments. The highest average was at the 15 kr treatment (8.96 %) and the lowest one was at 5 kr (6.50 %).The effects of the salinity treatments were significant. The highest average was at the 100 mM (8.50 %) and the lowest one was at 0 mM (control) treatment (7.10 %). The 15kr with 100 mM and 15 kr with 150 mM treatment   had the highest total carbohydrate content( 9.50 %). The lowest average  was  at the treatment  of  5 kr with  0 mM (4.70 %).These results were similar to those reported by Rashad (1995) on Tagest erecta,  El-Sharnouby et al. (1997) on  Hibiscus sabdariffia and Farid et al. (1999)  on  the sweet marjoram. The results of the other workers are not in harmony Kandeel et al. (1991) reported on Ocmium basilicum that the high gamma dose of  12000 r caused a slight decrease in comparison with the control.These results were similar to those reported by Zidan and Alzahrani (1994) on Ocmium basilicum.

The obtained results of salinity   treatments during the M₂-generation    of the  second season indicated that the  treatments of 100 and 150mM increased the amount of carbohydrate contents and  the increase was  significant at the treatment of 100mM compared  with the control. Many plants, which are stressed ,by Na Cl  salinity, accumulated starch and  soluble carbohydrates (Greenway and Munns,1980 and Rathert,1984). This   accumulation has  been attributed to impaired  carbohydrate utilization (Munns and Termaat,1986). Dhanapackiam and Ityas (2010) reported that the soluble and total carbohydrates content in leaves were higher in salt stress plants compared with the control. This is strong evidence that photosynthesis is the main source of accumulating carbohydrates under water stress.  The accumulation  of organic  solutes(soluble and  insoluble carbohydrates) might play  an important role in increasing  the  internal osmotic pressure ( Zidan  and Alzahrani,(1994). This has been widely regarded as response to salinity stress condition. Munns (1993) reported that the concentration of sugars and reserve polysaccharides always rise after plants are exposed to salinity in both growing and fully expanded tissues. This is consistent with  a blockage in utilization of  sugars in the growing tissues and a subsequent build-up in the rest of the plant.

     Table  (4). Average   values of the total carbohydrates content of Catharanthus roseus, L.as affected by gamma radiation (kr) and salinity levels (mM) treatments in the M₁ – M₂ generation of second season.¹⁾.

¹⁾Values marked with the same alphabetical letters, within comparable group of  means, do not differ   significantly, using

L.S.D. at 0.05 level of probability.