Collage for Girls, Al-Azhr University, Cairo Egypt
Application of In Vitro Technique to Produce
Safflower (Carthamus tinctorius L.) Plants Tolerant to Drought and Salinity Stress and Achievement
of α- tacopherol accumulation. H. S. Taha1, A. M. AbdEl-Kawy2 and A. A. Ezz-El-Din3
Plant Biotechnology Department, National Research Centre, Dokki, Cairo Egypt.1
Biology Department, Faculty of Science, Jazan University, KSA.2
Medicinal &Aromatic Plants Department, National Research Centre, Dokki, Cairo, Egypt.3
A successful protocol for in vitro production of safflower (Carthamus tinctorius L.) plants tolerant to drought and salinity stresses was established. The maximum percentage of calli induction was obtained on MS medium supplemented with 1.5 mg/l NAA and 0.5 mg/l BAp from immature embryo, leaf, cotyledon explants, respectively. However, supplementation of MS medium with 0.1 mg/l TDZ and 0.1 mg/l NAA enhanced direct shoots regeneration from different explants. In addition, fortifying MS medium with 0.3 mg/l 2iP achieved better shoots multiplication from immature embryo explants as compared to other explants. On the other hand, augmentation of MS- medium with 0.1 mg/l NAA increased the rate of rootlet shoots formation. The immature embryos were also subjected to saline or drought media for calli, regeneration and direct shoots multiplication. The saline MS-media contained 75,150,225 or 300 mM of NaCl. However, the MS-drought media were supplemented with 1, 2 or 3 % of PEG. The best results of calli, regeneration and direct shoots multiplication, fresh, dry weights (mg/jar) and percentage water content resulted from the MS-medium supplemented with 225 mM of NaCl as compared with the drought stress with 2% of PEG. Evaluation and determination of α tacopherol (vitamin E( in different type of cell lines was carried out using spectrophotometer. vitamin E is essential vitamin for human nutrition.
Safflower (Carthamus tinctorius L., Asteraceae) is an important oil seed crop cultivated throughout the semiarid regions of the world for its high content of linoleic acid (78% of total fatty acids) (Hojati et al. 2010). It is an important annual industrial crop. The stem, leaves, seeds and flowers are used for different purposes (Vedat et al., 2011). Safflower seeds contain 13 to 46% oil, and approximately 90% of this oil is composed of unsaturated fatty acids, namely oleic and linoleic acids (Johnson et al., 1999). Historically, safflo-wer was grown in Egypt and Euphrates exclusively as a source of red dye ‘cartha-min’ extracted from its florets. Around mid last century, its cultivation was exten-ded to Asia, Europe, Australia, and the Americas due to its recognition as a source of good quality oil valued for edible and industrial purposes. Safflower contains α-tocopherol, which accounts for more than 95% of the total tocopherols (α, β, γ, and δ; (Furuya et al. 1987). The oil from the seeds of safflower is a premium edible oil since its consumption help to lower blood cholesterol, as α-tocopherol is an efficient scavenger of activated oxygen species (Velasco et al. 2005). Besides, safflower also contains red (water insoluble) and yellow (water-soluble) pigments utilized for producing herbal medicines, food colorants, cosmetics, textile, and natural dyes (Fatahi et al. 2008; Li et al. 2009). Plant tissue culture plays an important role in the production and improvement of plants and in the manipulation of plants. Both in vitro culture of plant cells and tissue have attracted considerable attention as means for studying plant physiological and genetic processes as well as assisting in the breeding of improved cultivars through increasing genetic variability. It is expe-cted that regenerated plants have the same genotype as the mother plant; however, in some cases somaclonal variants have been found among regenerated plants (Wani et al., 2010). Moreover, the use of cell and tissue culture methods as means of producing medicinal metabolites is practiced a long time ago (Rout et al., 2000; Verpoorte et al., 2002). Since plant cell and tissue culture emerged as a discipline wit-hin plant biology, researchers have endeavored to utilize plant cell biosynthetic capabilities for obtaining useful products as well as for studying the metabolism (Misawa, 1994; Verpoorte et al., 2002).
Plants regularly face stressed growth conditions, such as drought, salinity, chilling, freezing, and high temperatures. The-se stresses can delay growth and development, reduce productivity, and, in extreme cases, cause plant death. Plant stress responses are dynamic and involve complex interactions between different regulatory levels, including metabolism adjustment and gene expression for physiological and morphological adaptation (Julia and Claudia, 2012).
Both drought and salinity are the most important abiotic stress which leads to increasing yield losses in crops in the arid and semi arid parts of the world (Fulda et al., 2011). There are different mechanisms by which plants showed with drought and salt stress. Therefore, it is necessary to study the physiological responses of crop plants to these stresses in order to develop appropriate strategies to sustain food production under adverse environmental conditions.
The aim of this study is to determine the morphological responses of safflower plants to salt and drought stress in their callus production, direct shoots regeneration and shoots multiplication as well as the evaluation and determination of α–tocopherol content.
2.MATERIALS AND METHODS
Safflower seeds were secured from the Horticulture Division, Faculty of Agriculture, Cairo University, Giza, Egypt.
2.2.1.Establishment of sterilized safflow-er seedling
Seeds of safflower were surface sterilized by immersing in 70% ethanol for 10 sec., followed by three washes with sterile distilled water. Then, the seeds were immersed in 20% of commercial Clorox (5.25 Cl g/l) solution containing a drop of Tween 20 for 15 min. The sterilized seeds were germinated aseptically on 50 ml of solidified basal MS-medium (Murashige and Skoog, 1962) in 300 ml glass jars. Ten replicates, each of 10 seeds per jar were used. Agar was added prior to autoclaving at 1.2 kg/cm2 for 20 min. The pH of the medium was adjusted to 5.8 by the addition of 0.1 N HCL or 0.1 N KOH. The jars were incubated in a growth chamber at 26 ± 1C and exposed to 16 h/day photoperiod at intensity of 3000 Lux from white cool light of fluorescent lamps.
2.2.2.Establisment of safflower callus cultures
Aseptically grown seedlings after 28 days from invitro germinated seeds on basal MS-medium were used as sources of different explants i.e. cotyledons, and leaf explants as well as immature embryos which were excised from sterilized immature seeds of safflower. Three sections 3-4 mm in diameter of leaf and cotyledons were excised and cultured in 150 ml of glass jars containing 25 ml of the following modified MS-medium:
1-MS free growth regulators
2-MS + 0.5 NAA
3-MS + 0.5 BAp
4-MS + 0.5 NAA + 0.5 BAp
5-MS + 1.0 NAA + 0.5 BAp
6-MS + 1.5 NAA + 0.5 BAp
7- MS + 2.0 NAA + 0.5 BAp
Where: naphthalene acetic acid (NAA) was used as auxin and 6-benzylamino purine
(BAP) was used as the cytokinin. 2.2.3.Direct shoots regeneration
An experiment was designed to study the efficiency of modified MS medium with NAA and TDZ (Thidiazuron) at different concentrations on direct shoots regeneration from immature embryos, cotyledons and leaf explants of safflower. Explants were cultured on the following MS medium:
MS free growth regulators
MS + 0.1 NAA
MS + 0.05 TDZ + 0.1 NAA
MS + 0.1 TDZ + 0.1 NAA
MS + 0.3 TDZ + 0.1 NAA
The following parameters were recor-ded as follow:
1- Percentage of direct shoots regeneration
2- Number of shoots/jar
3 shoot length (cm).
4-Fresh and dry weights(g/jar).
2.2.4. Shoots multiplication
An experiment was carried out to eva-luate the influence of MS medium supplemented with different concentrations (0.1,0.2,0.5,and 1mg/l) of 2iP (N6-[2-Iso-pentyl]adenine) on the achievement and enhancement of in vitro shoots multiplication of safflower. Then, the percentage of safflower shoots multiplication was reco-rded.
For rootlet formation of shootlets, the resulted multiple shootlets from immature embryos subjected to MS medium incorporated with different concentrations of NAA (0.1,0.3, 0.5,1) and free growth were used. The percentage of rootlets shoots formation was recorded as a parameter for in vitro roots development.
2.2.6. Effect of different tolerance of salinity or drought stresses on in vitro safflower morphological responses
In this experiment the best modified MS-media for either callus production, direct shoots regeneration or multiplication process was treated with different concentrations (75,150,225 and 300mM) of NaCl (sodium chloride) as saline stress or (1,2 and 3%) of PEG (polyethylene glycol) as drought stress. Saline or drought experiments were then carried out to investigate their efficiency on percentages of callus formation ; direct shoots regeneration and shoots multiplication. Moreover, fresh and dry weights (g/jar) and dry matter content (percentage) of resulted shoots was recorded.
2.2.7. Determination of α-Tocopherol content
The α-Tocopherol content was assay-ed as described by Backer et al. (1980) where five hundred milligrams of fresh tissue were homogenized with 10 ml of a mixture of petroleum ether and ethanol (2:1.6, v/v). Then the extract was centrifuged at 10,000 rpm for 20 min and the supernatant was used for estimating α-Tocopherol. To one ml of the extract, 0.2 ml of 2% 2,2-dipyridyl in ethanol was added and mixed thoroughly and kept in dark for 5 min. Resulting red color was diluted with 4ml of distilled water and mixed well. The resulting color in the aqueous layer was measured at 520 nm. The α-Tocopherol content was calculated using a standard graph made with known amount of α-Tocopherol and expressed in mg g−1 fresh weight (FW).
Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). The values are mean ±S.D. for five samples in each group. p values ≤0.05 were considered as significant.
3.RESULTS AND DISCUSSION
3.1.Establishment of safflower callus cultures
In this regard, as shown in Table (1) and Fig.(1) MS medium supplemented with different concentrations of NAA al-one or in combinations with 0.5 mg/l of BA were used to study their efficiency on the production of callus cultures from immature embryos, leaf and cotyledon explants of safflower. Cultures were incubated under light condition (16/8h) at 26± 1ºC for 28 days. The highest frequency of callus production; fresh and dry weights 96.7(%) 2.17 and 0.15(g/jar) were record-ed with MS medium supplemented with 1.5 mg/l NAA+0.5 mg/l Bap, respectively. The immature embryos explants showed high significance for callus production as compared to leaf and cotyledon explants, respectively. It is well documented that there are different factors affecting callus production from different explants, such as nutrients concentration, stress factors, light, incubation period, type, and concentration of growth regulators and were found to be important determinants of sec-ondary metabolite production. This work reveals that callus proliferation of safflower incubated for 28 days improved significantly on MS medium suppleme-nted with cytokinin (0.5 mg/l BA) in combination with auxins (1.5 mg/l NAA) along with optimum concentration of oth-er nutrients and growth regulators. These observations are in agreement with Smita et al. (2011) who reported that cell cultures of Carthamus tinctorius was improved significantly on MS liquid medium containing 50.0 μM (NAA) and 2.5 μM (BA) at 28 days of incubation period. Mo-reover and in similar reports for growth of calluses and cell cultures of C. tinctorius (Hanagata et al. 1992; Rani et al. 1996; Nikam and Shitole 1999; Gao et al. 2000).
Fig.(1). Callus production from immature embryos(1) leaf(2) and cotyledon (3) explants Carthamus tinctorius. Cultures incubated under light (16/8 h) condition at 26 ±1 ºC for 28 days.
3.2.Direct shoots regeneration
Data presented in Table (2-a and b) and Fig. (2) clearly reveal that the effect of fortified MS medium with auxins (NAA) at 0.1 mg/l alone or in combinations with different concentrations (0.05, 0.1 and 0.3 mg/l) of cytokinin (TDZ) on in vitro direct shoots regeneration from immature embryos, leaf and cotyledon explants. It should be mentioned that the maximum percentage of direct shoots regeneration, number of shoots/jar, highest shoot length (Table 2-a) and fresh and dry weights g/jar (Table 2-b) recorded 87.6, 8.2, 2.25 cm, 1.65 and 0.025 (g/jar) were recorded with direct shoots regeneration derived from immature embryos, respectively. Moreover, augmentation of MS medium with 0.1 mg/l of auxins (NAA) in combination with 0.1mg/l of cytokinin (TDZ) was the best for enhancing the process of direct shoots regeneration from the used different explants compared with other concentrations of cytokinin (TDZ).
Fig. (2). Direct shoots regeneration from immature embryos explants of Carthamus tinctorius. Cultures incubated under light (16/8 h) condition at 26 ±1 ºC for 28 days. 3.3Shoots multiplication
Data in Table (3) and Fig.(3) present the effect of fortified MS medium with different concentrations of 2iP on achiev-ement of in vitro direct shoots multiplication of safflower. The highest percentage of direct shoots multiplication rate (96.7 shoots/28 days) was recorded with MS medium supplemented with 0.3 mg/l 2iP as compared with the other supplementations. Similarly, Sujatha and Dinesh kum-ar (2007) reported that Highly prolific adventitious shoot regeneration was obser-ved in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on the medium supplemented with TDZ + NAA. Other studies carried out by Sujatha and Suganya 1996 and Radhika et al. 2006 revealed positive influence of TDZ + NAA and BA + NAA on induction of caulogenic response from seedling explants of C. tinctorius. Moreover in an agreement with our obtained results Tere-sa and William (1993) reported that direct shoot regeneration from primary explants was optimal on MS medium containing 0.1 mg/l NAA and 0.5 mg/l BA or 0.1 mg/l of TDZ. They also reported that the highest shoots multiplication was observed with Ms medium incorporated with 1 mg/l of 2iP.
Table (3). Effect of fortified MS-medium with 2iP at different concentrations (ppm) on in vitro shoots multiplication percentage from immature embryos of Carthamus tinctorius. Cultures incubated under light (16/8 h) condition at 26 ±1ºC for 28 days.
MS medium supplemented with: (ppm)
MS free growth regulators
MS + 0.1 2iP
MS + 0.3 2iP
MS + 0.5 2iP
MS + 1.0 2iP
Values followed by different small letters in each column are significantly (P≤0.01) according to Duncan Multiple Range test.
Mean ± Standard Division (SE). Each mean is the average of five replicates.
Fig.(3). Direct shoots multiplication from immature embryos of Carthamus tinctorius. Cultures incubated under light (16/8 h) condition at 26 ±1ºC for 28 days. 3.4.Rootlets formation
For induction of rootlets on derived multiple shootlets from immature embryos; shoots were subjected to MS medium containing different concentrations of auxins (NAA), the maximum percentage of rootlets formation 98.6 was noticed with MS-medium supplemented with 0.1 mg/l NAA as compared with the other NAA concentrations. In this respect Tere-sa and William (1993) reported that in vitro derived shootlets were rooted on 0.5X of MS medium containing 1 mg/l of NAA.
3.5.Effect of different tolerant stress salinity or drought on in vitro safflower morphological responses
The effect of different environmental stresses such as salinity and drought on the different morphological responses: callus formation (%), direct shoots regeneration (%), shoots multiplication (%) shoots fresh and dry weights and dry mater content were investigated. Data displayed in Table (4) show that the highest percentage of callus formation (65.8%); direct shoots formation (59.6%); shoots multiplication (15.9%); shoots fresh (0.85 g/jar) and dry (0.07 g/jar) weights and dry matter content (8.23%) were recorded with MS medium fortified with 225 mM of NaCl as compared with other supplementations. However the supplementation of MS medium with 2(%) of PEG (Table 5) resulted 62.8(%) of callus formation; 45.9(%) of direct shoots formation; 11.4(%) of shoots multiplication; 0.65 (g/jar) shoots fresh and (0.07 g/jar) dry weights and dry matter content (8.23%) when compared with 1 or 3(%) of PEG. In this respect it should be mentioned that the treatment of MS medium with 225mM of NaCl recorded the best results of the different morphological responses compared with drought of MS medium with 2(%) of PEG.
3.6.Determination of α-Tocopherol content (µg/g F.W).
α-Tocopherolwas determined in callus (MS-medium supplemented with 1.5 mg/l NAA +0.5 mg/l BA), direct shoots regeneration (Ms-medium supplemented with 0.1 mg/l TDZ +0.1 mg/l NAA) and shoots multiplication (MS-medium supplemented with 0.3 mg/l 2iP) responded better than either modified MS medium (without any stress); modified MS medium supplemented with 225mM of NaCl or modified MS medium containing 2(%) PEG. Calli cultures, direct shoots regeneration or shoots multiplication were produced from immature embryo, leaf or cotyledon explants of safflower. Data in Fig.(4) clearly indicated that the highest values of α-Tocopherol content 112.3, 91.7 and 86.8 µg/g (F.W) were produced from shoots multiplication, direct shoots regeneration and calli cultures derived from immature embryos and cultured on modified MS medium augmented with 225 mM of NaCl, respectively. However, treating modified MS medium with 2% PEG recorded 95.9, 87.4 and 79.6 µg/g (F.W) for derived shoots multiplication, shoots regeneration and calli culture from immature embryos, respectively. Therefore, it may be inferred from these observations that the variation in yield of α-tocopherol might be due to the diversity and specificity associated with the interactions between plant cell receptors ad NaCl (as abiotic stress). Similar reports of an improved production of secondary metabolites have been reported for cell cultures of Xanthophyllomyces dendrorhous and Rubia tinctorum (Wang et al. 2006; Orban et al. 2008). Metal ions, such as sodium, magnesium, calcium, manganese, zinc, copper, iron, cobalt, etc., at appropriate concentrations could act as signaling molecules and trigger the biosynthesis of such valuable compounds in the stressed tissue of various plants (Savitha et al., 2006). Smita et al. (2011) reported similar results and indicated that NaCl and MgSO4 improved the production of α-tocopherol and red pigment and yellow pigment. NaCl is an effecter molecule responsible for maintenance of the osmotic balance of the cell. It also generates toxic reactive oxygen species (ROS). Therefore, to reduce the toxicity of ROS and yet maintain the osmotic balance, cells synth-esize of nonenyzmatic antioxidants, such as tocopherols, carotenoids, etc., besides certain enzymatic antioxidants is required (Hojati et al. 2010).
Moreover, Jung (2004) and Wang et al. (2009) reveal that when plants are subjected to environmental stresses such as drought, salinity, heat, chilling, and mineral deficiency, a variety of toxic, active oxygen species (AOS) such as superoxide (O-2), hydrogen peroxide (H2O2), hydroxyl radicals (OH), and singlet oxygen (O2) were observed. The balance between the production of AOS and the quenching activity of the antioxidants is upset and this often results in oxidative damage. However, Munne (2005) mentioned that environmental stresses trigger a wide variety of plant responses, ranging from altered gene expression to changes in cellular metabolism and growth. α-tocopherol which is the major vitamin E compound found in leaf chloroplasts, deactivates photosynthesis-derived reactive oxygen species (mainly 1O2 and OH), and prevents the propagation of lipid peroxidation by scavenging lipid peroxyl radicals in thylakoid membranes. The level of α-tocopherol changes differentially in response to environmental constraints, depending on the magnitude of the stress and species-sensitivity to the stress. Car-etto et al. (2002) stated that always more than 91% of the total tocopherols was represented in extracts from sunflower plantlets as well as in cell cultures. Most photosynthetic food plant tissues contain between 10 and 50 mg (g FW)1 tocopherols (Hess, 1993). Moreover Sofia et al. (2004) established a protocol for in vitro production system of natural a-tocopherol. They reported and in agreement to the obtained results that the most efficient medium was found to be MS basal medium with NAA and BA with the addition of casaminoacids and myo-inositol which improved to 30% when homogentisic acid was used. Also, Ramachandra et al. (2004) reported that among the environmental stresses, drought stress is one of the most adverse factors of plant growth and productivity. Understanding the biochemical and molecular responses to drought is essential for a holistic perception of plant resistance mechanisms to water-limited conditions. Drought stress progressively decreases CO2 assimilation rates due to reduced stomatal conductance. Drought stress also induces reduction in the contents and activities of photosynthetic carbon reduction enzymes, including the key enzyme, ribulose-1,5-bisphosphate carboxylase/oxyge-nase. The critical roles of proline and glycine-betaine, as well as the role of abscisic acid (ABA), under drought stress conditions have been actively researched to understand the tolerance of plants to dehydration. In addition, drought stress-induc-ed generation of active oxygen species is well recognized at the cellular level and is tightly controlled at both the production and consumption levels in vivo, through increased antioxidative systems. Knowledge of sensing and signaling pathways, including ABA-mediated changes in response to drought stress, is essential to improve crop management. AS Mostafavi (2011) reported that salt stress adversely affected the germination percentage, germination rate, shoot length, root length, seedling length, and root to shoot length ratio, seed vigour, and germi-nation index and mean germination time of all 6 genotypes of safflower and a significant variation in salt tolerance was observed among all the safflower.
In general it can be concluded that culturing of immature embryos of safflower as explants on MS-medium supplemented with either 1.5 mg/ NAA + 0.5 mg/l BA or 0.1 mg/l TDZ +0.1 mg/l NAA or 0.3 mg/l 2iP in presence of 225 mM of NaCl or PEG at 2 (%) can produce calli cultures, plantlets via direct regeneration or direct shoots regeneration tolerant to saline or drought nutrient-medium, respectively.
Fig. (4). Effect of modified MS medium on accumulation of α-Tocopherol (µg/g FW) in calli, direct shoot regeneration and shoots multiplication produced from immature embryos, leaf and cotyledon cultures of Carthamus tinctorius. Cultures incubated under light (16/8 h) condition at 26 ±1ºC for 28 days. Table (4). Effect of augmented modified MS-medium with NaCl at different concentrations (mM) on callus formation (%), regeneration (%), shoots multiplication (%), shoots fresh and dry weights (g/jar) and moisture (%) from immature embryos of Carthamus tinctorius. Cultures incubated under light (16/8 h) condition at 26 ±1ºC for 28 days.
NaCl concentrations (mM)
Callus formation (%)
Direct shoots regeneration (%)
Shoots multiplication (%)
Shoots fresh weight (g/jar)
Shoots Dry weight (g/jar)
Shoots dry matter content (%)
Mean ± Standard Division (SE). Each mean is the average of five replicates.
Table (5). Effect of fortified modified MS-medium with PEG at different concentrations (%) on callus formation (%), regeneration (%), shoots multiplication (%), shoots fresh and dry weights (g/jar) and moisture (%) from immature embryos of Carthamus tinctorius. Cultures incubated under light (16/8 h) condition at 26 ±1ºC for 28 days.
Callus formation (%)
Direct shoots regeneration (%)
Shoots multiplication (%)
Shoots fresh weight (g/jar)
Dry weight (g/jar)
Shoots dry matter content (%)
Mean ± Standard Division (SE). Each mean is the average of five replicates.
For: 1-Callus formation ;MS medium supplemented with 1.5 mg/l NAA + 0.5 mg/l BA.
2-Regeneration: MS medium supplemented with 0.1 mg/l TDZ + 0.1 mg/l NAA.
3- Multiplication: MS medium supplemented with 0.3 2iP.
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تطبيق التقنيات المعملية لإنتاج نباتات قرطم مقاومة للإجهاد المائى والملحى
حسين سيد طة1-عائشة محمد عبد القوى2-عزة أمين عزالدين3
قسم التكنولوجيا الحيوية النباتية - المركز القومى للبحوث - القاهرة - مصر 1
قسم البيولوجى - كلية العلوم - جامعة جيزان - المملكة العربية السعودية 2
قسم النباتات الطبية والعطرية - المركز القومى للبحوث - القاهرة - مصر 3
تم تاثيث برنامج متكامل لانتاج نباتات قرطم معمليا مقاومة للاجهاد المائى والملحى، حيث أنتجت بيئة موراشيج وسكوج المحتوية على 1.5 ملليجرام/لتر نفثالين حمض الخليك NAA و0.5 ملليجرام/لتر بنزيل امينو بيورين أعلى نسبة من الكالوس من الاجنة الغير ناضجة، الورقة، الأوراق الفلقية على الترتيب، و لكن أضافة 0.1 ملليجرام/لتر ثاداى ايزرون TDZ مع 0.1 ملليجرام/لتر من نفثالين حمض الخليك NAA نشطت تكوين النموات الخضرية، ولكن اضافة 0.3 ملليجرام/لتر من 2iP ايزوبنتينيل ادينين الى بيئة موراشيج وسكوج حفزت تضاعف النموات الخضرية من الأجنة الغير ناضجة مقارنة بالأجزاء النباتية الاخرى، و من ناحية أخرى وجد أن إضافة 0.1 ملليجرام/لتر نفثالين حمض الخليك NAA زادت من تكوين المجموع الجذرى على النموات الخضرية الناتجة، وكذلك قد تم دراسة استجابة الأجنة الغير ناضجة عند تعرضها لمجموعة من البيئات المغذية المحتوية على تركيزات مختلفة من الملوحة 75، 150، 225، أو 300 مللى مول من كلوريد الصوديوم (إجهاد ملحى)، أو بيئات مغذية محتوية على البولى ايثلين جليكول PEG (إجهاد مائى) بتركيزات 1،2،3(%)، وكانت أفضل النتائج المتحصل عليها بالنسبة لتكوين الكالوس وتكوين المجموعات الخضرية وتتضاعفها عند تعرضها للإجهاد الملحى بنسبة 225 مللى مول بالمقارنة بتعرضها للإجهاد المائى بنسبة 2% كما تم تقدير الفاتاكوفيرول (فيتامين E) باستخدام مطياف الكتلة الضوئى وهو فيتامين مهم وأساسى لتغذية الإنسان.
* العنوان الدائم: قسم النبات - كلية العلوم للبنات -جامعة الازهر- القاهرة -مصر
الكلمات المفتاحية: القرطم، كالوس، إعادة تكوين النموات الخضرية، التضاعف الخضرى، إجهاد مائى، إجهاد ملحى فيتامين.