GENETICAL STUDIES ON HAPLOID PRODUCTION IN SOME ORNAMENTAL

By MOHAMMED ADLY MOHAMMED MOSTAFA B. Sc. Agric. (Genetics), Al-Azhar University 1996 M. Sc. Agric. (Agric. Botany - Genetics), Al-Azhar University 2002

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree

Of DOCTOR OF PHILOSOPHY

In AGRICULTURAL SCIENCES (Agriculture Botany – Genetics)

Department of Agriculture Botany Faculty of Agriculture, Cairo Al-Azhar University

1434A.H. 2013 A.D. APPROVAL SHEET

NAME: MOHAMMED ADLY MOHAMMED MOSTAFA TITEL: GENETICAL STUDIES ON HAPLOID PRODUCTION IN SOME ORNAMENTAL

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree Of DOCTOR OF PHILOSOPHY

In AGRICULTURAL SCIENCES (Agriculture Botany – Genetics)

Department of Agriculture Botany Faculty of Agriculture Cairo Al-Azhar University 1434 A.H. 2013A.D. Approved by: Prof. Dr. ESSAM AHMED EL-SAYED RAGAB Prof. of National Center for Radiation Research and Technology. Prof. Dr. MOHAMED ALI ABD EL-RAHMAN Prof. of Genetics, Dept. of Agric. Botany, Fac. of Agric. Al-Azhar University, Cairo. Prof. Dr. ABD EL-HADI IBRAHIM HASSN SAYED Prof. of Genetics, Dept. of Agric. Botany, Fac. of Agric. Al-Azhar University, Cairo.

Date: / /2013

TITEL: GENETICAL STUDIES ON HAPLOID PRODUCTION IN SOME ORNAMENTAL PLANT

NAME: MOHAMMED ADLY MOHAMMED MOSTAFA

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree

Of DOCTOR OF PHILOSOPHY

In AGRICULTURAL SCIENCES (Agriculture Botany – Genetics)

Department of Agriculture Botany Faculty of Agriculture Cairo Al-Azhar University

1434 A.H. 2013 A.D.

Supervision Committee: Prof. Dr. ABD EL-HADI IBRAHIM HASSN SAYED Prof. of Genetics, Dept. of Agric. Botany, Fac. of Agric. Al-Azhar University, Cairo. Late Prof. Dr. ABD EL-AZIZ HASSNABD-ALLA Prof. of Genetics, Dept. of Agric. Botany, Fac. of Agric. Al-Azhar University, Cairo.. Prof.Dr. AYMAN ABD EL-MGEED EL-FIKI Prof.of Biotecnology, National Center for Radiation Research and Technology.

ACKNOWLEDGEMENT

With the name of ALLAH

I wish to express my deepest gratitude and sincere appreciation to Prof. Dr. Abd El-Hadi I. H. Sayed professor of Genetics, Agric. Botany department, Faculty of Agriculture, Al-Azhar University, for his continuous supervision, help, valuable assistance and suggestions, useful advice and guidance throughout this investigation.

I would like also to express my greet thanks to Late Prof. Dr. Abd El-Aziz H. A. Mohamed professor of Genetics, Agric. Botany department, faculty of Agriculture Al-Azhar University for his suggesting the problem.

I am greatly thanks to Prof. Dr. Ayman A. El-Fiki professor of Biotechnology, National Center for Radiation and Technology for his asstiance supervision, suggestion, greet help, encouragement during this study.

Grateful thanks are also extended to all staff members genetics in each colleagues of department Botany; Agric. Fac.; Al-Azhar Univ. and Natural Production National center for Radiation Research and Technology, for their encouragement, cooperation and friendly attitude.

Content Page Introduction 1 Review of Literature 4 I- Tissue Culture 4 a-Induction of haploid 9 b. Regeneration 10 c- Diplodization. 15 II. Radiation effect on haploid plant induction 16 III.Adaptation 22 IV. Haploid plants under Salinity stress 24 V. Chemical Analysis 27 -Proline and Protein contents 27 V. Molecular Markers 3 0 1- SDS- PAGE electrophoresis 30 2-Randomly amplified polymorphic DNA (RAPD) 33 MATERIAL AND METHODS 37 1-MATERIALS 37 A. Plant material 37 B. Gamma irradiation source 37 C-Media 37 D-Callus Culture Media 39 a) Callus induction media 39 b) Regeneration media 39 METHODS: 39 Callus subculturing and haploid induction; 39 B- Colchicine treatments 40 C-Gamma irradiation treatment 40 D- Salinity treatments 40 E- Combined effect between gamma radiation and salinity 40 F-Adaptation 41 Chemical analysis 42 Estimation of proline content 42 Estimation of Protein content 42 Molecular Markers 4 2 a- SDS- PAGE electrophoresis 42 b-Randomly amplified polymorphic DNA (RAPD) 47 RESULT AND DISCUSSION 49 I- Induction of haploid anther callus 49 II-Haploid anther callus Induction 55 III-Regeneration of haploid plants 55 IV-The haploid confirmation 61 V-Diplodization of haploid plants 63 VI- Radiation effect on haploid anther callus induction and regeneration 66 VII- Radiation effect on haploid plant induction 68 VIII- Haploid plants under Salinity stress 76 IX- The gamma radiation and NaCl combined effect on haploid plantlets of alata. 80 X- The proline contents; Biochemical Analysis in haploid plants 85 a-Under Radiation stress 85 b-Under salt stress 86

II c- The combined effects between gamma radiation doses and NaCl concentrations on proline contents in haploid N. alata plants 86 B- Total protein a) Under radiation stress 91 b-Under salt stress c- The combined effects between gamma radiation and salinity on total protein 95 C-Molecular Markers 98

1- SDS- PAGE electrophoresis 98 a-The effect of ploidy level on protein bands 98 b- Effect of gamma radiation doses on SDS-PAGE 98 banding pattern of Nicotiana alata plantlets

C-Effect of NaCl concentrations on protein banding patterns in Nicotiana alata plantlets 102 d) The combined effects between gamma radiation doses and salinity on protein banding pattern 105 2- Randomly amplified polymorphic DNA markers (RAPD) 117 SUMMARY 133 REFERANCES 137 ARABIC SUMMARY

III

Haploids are plants with a gametophytic chromosome number and doubled haploids are dihaploids that have undergone chromosome duplication. The production of haploids and doubled haploids (DHs) through gametic embryogenesis allows a single-step development of complete homozygous lines from heterozygous parents, shortening the time required to produce homozygous plants in comparison with the conventional breeding methods that employ several generations of selfing. The production of haploids and DHs provides a particularly attractive biotechnological tool, and the development of haploidy technology and protocols to produce homozygous plants has had a significant impact on agricultural systems. Nowadays, these biotechnologies represent an integral part of the breeding programmes of many agronomically important crops. There are several available methods to obtain haploids and DHs, of which in vitro anther or isolated microspore culture are the most effective and widely used (Germana Maria 2011). Tissue culture techniques, particularly short-term culture procedures such as shoot-tip culture and regeneration from primary explants, have been proposed as methods for obtaining large numbers of plants identical to the plant used as an explant source( Evans et al., 1984). Nicotiana spp. are one of the most important commercial crops in the world ( Liu and Zhang, 2008). Nicotiana alata is member from family solanacea, it is ornamental plant and the diploid cells contains18 chromosomes. Nitsch (1969) reported the first production of haploid plants through anther culture and regeneration of plants of Nicotiana alata, For these reasons they have been considered to suitable candidates for model species in somatic cell genetics research( Bourgin et al., 1979). Radiobiological studies on plant tissues in culture may provide information on the cell growth behavior, radiosensitivty and the induction of mutations. The radiosensitivity of plants and calli can be manifested mostly in three ways: 1) inhibition

1 of growth, 2) reduction of reproduction capacity and 3) death. growth inhibition induced by ionizing radiation has been attributed to chromosome deletion (Sparrow et al. 1961) and changes in a variety of biochemical and physiological systems Gunckel and Sparrow, (1961). Irradiation with gamma rays may provide and insight into the mechanism of action of radiation in producing physiological and genetic variability, thus it has been directly used to produce useful variation in quantitatively inherited characters, such as quality and maturity time (Brock, 1970). Salinity is a major factor limiting the crop productivity in the semi arid area of the world (Robinson, 1986). Salinity inhibits plant growth by one or more of the three principle ways; 1st. ion toxicity (mainly of Na+ and Cl-), 2nd. osmotic stress and the 3rd. nutritional disruption (Yeo et al., 1991). These include genetic variability between species, or among within species and duration and timing of exposure to salinity (Cushman et al. 1990) they added that salt tolerance of halophytes depends on the constitutive expression of several genes in response to salt stress. The application of mutagens in tissue culture in vitro to enhance the rates of spontaneous mutations and the use of direct selection for the screening of spontaneous mutants or variant lines, have been used in several laboratories. In addition, an increase in genetic variability may be induced in large and homogeneous populations of plant cells or in callus tissues, by exposing cultures to physical or chemical mutagenic agents. These are capable of increasing the frequency of changes in the genetic material when the cultures are under conditions which allow for the rapid screening of the mutants. These conditions may also be used to select and recover spontaneous variants or mutants directly from untreated material. The main types of

2 mutants are (1) auxotrophic, which require nutritional supplements for normal growth, (2) resistant, which resist specific drugs, antimetabolites, or abnormal environmental or nutritional condition and (3) autotrophic mutants, that grow in deficient media and are capable of synthesizing some substances normally required for wild lines. The use of mutagenesis and in vitro induce mutation should provide a new input to the solution if breeding problems in crop plant (Ancora and Sonnino, 1987). The aim of the present investigation is production of haploid plants through anther culture in N. alata and study the effect of gamma rays, salt stress and combined effect between them on it. Morever, determining the changes in the internal composition of the cell as proline and protein. Also, to study the molecure diversity as revealed by; Protein Acrylamid Gel Eelectrophoresis - SDS (PAGE-SDS), Random Amplified Polymerphic DNA (RAPD) .

3 Review

Nicotiana spp. is one of the most important commercial crops in the world. Nicotiana alata is as a diploid plant; werwase its cells containe (18) chromosomes (Liu and Zhang, 2008).The family is composed of 98 genera and about 2300 species belonging to 14 tribes grouped in three sub families (D'Arcy, 1991). The Nicotiana consists of 64 recognized species, some species are cultivated as drug or ornamental plants (Roju, et al., 2009).

Tissue Culture Love and Rhodes (1985) cited in sweet potato that the basic medium used employs the salt base of Murashige& Skoog (1962) with 0.7% agar. An increase in sucrose concentration from 30 to 50 g/liter increased meristem survival, frequency and speed of regeneration. Optimum growth regulator concentrations, over a wide range of genotypes, were 0.3 mg/liter BA and 0.03 mg/liter NAA. Response to BA concentration was highly genotype dependent. Transfer to a medium without growth regulators following the formation of shoot primordia accelerated regeneration. The optimum pH for regeneration was nearer 5.2 than the standard 5.7. Murata et al (1989) found that the sweet potato meristem tips were cultured on MS medium supplemented with various growth regulators. Best results were obtained with the variety Kokei 14 on medium containing 2 mg NAA and 1 mg benzyladenine (BA) or 0.2 mg NAA and 2 mg BA/litre. Plantlets developed within 90 days. The variety Koganesengan was cultured on MS medium containing 0.2 mg NAA and 1 mg BA/litre. Sucrose was more effective than glucose as a carbon source and the optimal concentration was 30 g/liter. Santangelo and sonnino (1991) cited the work with 5 varieties of sweet potato demonstrated the possibility of propagating the crop not only in vitro but also in vivo via stem cuttings. There were vareital differences in capacity for

4 propagation of the 2 media tested for in vitro micropropagation, that without of the benzyladenine was superior to that with 0.3 mg /litre. Rosu and Ciofu (1995) showed that the effective micropropagation method using apical meristem culture of sweet potato was devised on the basis of tests to determine the effect of various explant sources, apex sizes supplements to the basal MS medium and genotypes on shoot proliferation. The growth of plants obtained in vitro was comparable to that of plants obtained by the traditional method of cloning using rooted cuttings. Kong et al (1998) Studied the culture of apical meristems of 4 sweet potato cultivars on MS medium supplemented with BA [benzyladenine], IAA and GA3 [gibberellic acid] resulted in 68.1-86. 6% plantlets production within 46-64 days. The best time for transplanting plantlets onto a second medium was 18-21 days. MS medium supplemented with NAA would be better for the rapid propagation of virus-free seedlings of sweet potatoes. When vermiculite was used as the transplant medium, the survival rate of virus-free plantlets could reach 100%. Baruah et al (1996) concluded that BAP was more superior than Kin for shoot proliferation of all studied citrus species. The best shoot proliferation was observed in the presence of BAP at 0.5 mg/l for C. assamensis, C. latipes and C. indica. Chen (1995) illastrated that plantlets were obtained from citrus pollen on improved MS medium containing 0.25 mg BA and 0.1 mg IAA /l. Starrantino and Caponnetto, (1990) stated that development of ovules of 5 orange cultivars succeeded on Murashige and Tuker medium (MT) with or without BA and or kinetin. Wu et al., (1990) suggested that somatic embryos and embryogenic callus were developed on MT medium without growth regulators when the ovules of the mandarin cv. Hunongbendizo, which produced a high proportion of mono embryonic seeds, were used. Rakleviciene and Sauliene, (2001) stated that the induction of morphogenesis with exogenously applied

5 phytohormones was studied in flowering Nicotiana alata flower stalk thin layer tissues under a long-day (16 h/d light) and short- day (8 h/d light) photoperiod, as well as in non-flowering N. tabacum discs under 16 h/d light and dark conditions. After phytohormonal treatment, cultivation was continued on a medium with no phytohormones. The same method of treatment was used in experiments performed with Asparagus officinalis callus. The yield of flowers, vegetative buds, and embryos was estimated after 30 and 45 days from the beginning of the experiment for tobacco and asparagus, respectively. The number of organs and embryos in tissues grown on a medium without or with phytohormones in the course of the whole experiment was also evaluated. Under different day to night ratios, the appearance of primary vegetative buds and shoots in N. alata flower stalk thin layer tissues was determined after 3 days of contact with auxin- (1 micro M indole-3-acetic acid; IAA) and cytokinin- (1 micro M 6-benzylaminopurine [benzyladenine]; BA) supplemented medium. Only vegetative buds appeared under a short day, and their number per explant was larger (from 22 to 28 buds per explant) compared with the total vegetative bud and flower number under long-day conditions (from 14 to 18 buds per explant). The induction of flower formation reduced the vegetative course of morphogenesis, but more intensive growth of bud and shoot biomass was observed. Direct somatic embryogenesis was observed in N. tabacum leaf discs after a 10- day initiation with 1 micro M BA, and the maximum (77.17± 5.65 somatic embryos (SE) per explant) was reached after 14 days of phytohormonal treatment under 16 h/d. The elimination of the phytohormonal stimulus after embryogenesis resulted in an increase in the number of embryos. Darkness retarded the induction of somatic embryogenesis and decreased the morphogenetic potential of the investigated explants. A similar effect of phytohormones was not seen in embryogenic callus of asparagus. In contrast to the action of auxin 2.4- dichlorophenoxyacetic acid, the highest level of embryogenesis was observed when callus was cultivated for 45 days on MS

6 supplemented with cytokinin BA (29.27 ± 3.02 SE per 100 mg embryogenic callus).

Sauliene and Rakleviciene (2002) showed that the phytohormonal control of the transition to de novo morphogenesis was demonstrated in cultures of thin layer tissues obtained from stem and leaf discs of non-blooming and from flower stalk tissues of blooming Nicotiana alata. Neither organs nor somatic embryos occurred when explants were grown on a medium supplemented with 1 micro M auxin (IAA) in the absence of cytokinin (BA). Only vegetative buds formed in non-flowering tobacco stem thin layer tissues irrespective of auxin-to-cytokinin ratio. BA used in four concentrations (1, 2.5, 5, and 10 micro M) with the amount of the auxin constant, appeared to be an important effect or of both organogenesis and somatic embryogenesis. Vegetative organogenesis in stem thin layer tissues of non-blooming plants was most significant at 2.5 and 5 micro M BA. However, inhibition of the vegetative and promotion of the generative development were observed under the same conditions in flower stalk thin layer tissues of blooming Nicotiana. The realization of the flowering stimulus in isolated flower stalk tissues was considerably dependent on BA at 1-5 micro M. An increase of BA up to 10 micro M greatly stimulated the vegetative development and inhibited the formation of reproductive organs. The elimination of IAA from the media of leaf explant cultivation revealed the effectiveness of BA in direct somatic embryogenesis. The data confirmed the fact that both vegetative organ neoformation and reproductive development significantly depend on the amount of BA when the floral stimulus in inflorescence tissues is realized and somatic embryogenesis is performed only at certain BA concentrations.

Sanatombi and Sharma (2007) concluded that the efficient micropropagation protocol was developed for Capsicum annuum cv. Morok Amuba, an ornamental chilli using shoo-tip and axillary shoot-tip explants. Multiple

7 shoot buds were induced from shoot-tip explants on MS medium containing cytokinins alone or in combination with IAA. A maximum number of shoot buds was induced on MS medium containing 10 mg Zeatin /litre followed by 5 mg BAP/litre in combination with 1 mg IAA/litre. Rooting and elongation of the shoot buds were achieved on MS medium supplemented with 0.5 mg/litre of IAA or IBA. Axillary shoots were induced on the rooted plantlets by decapitation and the axillary shoot-tips explants were used for further induction of shoot buds by culturing them on a medium containing combinations of BAP with IAA. The shoot buds were rooted on a medium containing 0.5 mg IBA/litre. The plantlets showed 80-90% survival during transplantation.

Koleva-Gudeva et al. (2007) suggested that the frequency of obtained androgenic plants depends highly on the genotype, therefore the low rate of haploid recovery limits the utility of anther culture in pepper breeding. The need for incubation treatment and adequate nutrition media supplemented with plant growth regulators, especially auxins, are suggested as important factors to obtain somatic haploid embryos in Pepper anther culture. The effect of three incubation treatments of the androgenic potential in pepper anther culture on MS, Nitsch (N), LS (Linsmaer and Skoog, 1965), NN (Nitch and Nitch, 1969) and CP medium are summarized, and the results demonstrate that by incubating treatment in cold conditions (at 7 °C) in darkness for 7 days, and then transferring the explants to light conditions (12-h photoperiod at 25 °C) for 4 weeks, on LS and NN mediums, anthers produced callus by incubating treatment in heat conditions (at 25 °C) in darkness for 7 days, and then transferring the explants to light conditions (12-h photoperiod at 25 °C) for 4 weeks, on MS and N mediums, anthers produced callus; by incubating treatment in heat conditions (at 35 °C) in darkness for 8 days, the next 4 days to light conditions (12-h photoperiod at 25 °C) on CP medium, and then transferring the explants to R1 medium for 4 weeks, anthers produced embryos.

8 Induction of haploid Haploid is a general term that refers to a plant containing the gametophytic number of chromosomes, that is, a single set of chromosomes in the sporophyte. Although in some special cases individual workers obtained haploids spontaneously or experimentally by special methods, until 1964 the large scale production of haploids in higher plants was only a theoretical possibility. In 1964, Guha and Maheshwari reported direct development of embryos from microspores of Datura innoxia by the culture of excised anthers. Anther culture provides a quick route in obtaining pure lines in a single generation from either green haploid plant that are artificially or spontaneously doubled. Indica rice is known as recalcitrant genotypes because it is difficult to obtain sufficient number of green plantlets of the regenerated plants from anther cultures. Anther culture research needs to be done to enhance rice breeding program Iswari et al. (2009) Haploids occur spontaneously at a low frequency, or they can be induced by several methods, such as modified pollination methods in vivo (wide hybridization, chromosome elimination, pollination with irradiated pollen, etc.) and by in vitro culture of immature gametophytes. Gametic embryogenesis is one of the different routes of embryogenesis present in the plant kingdom, and it consists in the capacity of male (microspore or immature pollen grain) or female (gynogenesis) gametophytes to irreversibly switch from their gametophytic pathway of development towards a sporophytic one. Differently from somatic embryogenesis, which provides the clonal propagation of the genotype (unless the somaclonal variation), gametic embryogenesis results in haploid plants (unless spontaneous or induced chromosome duplication occurs), because such plants are derived from the regeneration of gametes, products of meiotic segregation. Microspore or pollen embryogenesis (also referred to as androgenesis) is regarded as one of the most striking examples of cellular totipotency (Reynolds, 1997), but also as a form of atavism. It is an important survival adaptation mechanism in the plant kingdom that is expressed only under

9 certain circumstances and as a consequence of an environmental stress (Bonet et al., 1998). The production of haploids through gametic embryogenesis for breeding purposes has been studied by many research groups since the 1970s. There are many published reviews on the production of haploids and dehaploids (DHs), including those of (Dunwell, 2010). The DH techniques have been well established in a range of economically important crop species, including major cereals and cabbages (Wedzony et al. 2009). Gynogenesis is the least favoured technique at the present time because of its low efficiency, but it has been applied to species that do not respond to more efficient methods (Forster et al., 2007). The ability to obtain haploids and DHs is one of the most important applications of pollen biotechnology in plant breeding and genetics, involving the manipulation and reprogramming of pollen development and function (Testillano et al., 2000).

Regeneration Haploid callus were regenerated by several workers, method used were including; anther, buds, , stems or roots calturing. Liu and Cantliffe (1984) illustrated that the leaf, shoot- tip, stem and root explants gave rise to two types of callus on nutrient agar medium containing 0.5 to 2.0 mg/litre 2,4-D. The callus type which was compact, organized and bright to pale yellow gave rise to numerous globular and heart-shaped embryoids which regenerated into plantlets on transfer to hormone-free medium. The plantlets were transplanted to soil where they flowered and former storage roots at maturity. Rey and Mroginski (1985) showed that the series of in vitro tissue cultures, apical meristems 0.4-0.6 mm long of sweet potatoes cv. Colorada comun and Morada INTA were grown in Murashige and Skoog nutrient solution supplemented with various concentration of IAA, NAA, kinetin or GA3. Addition of 0.1 mg NAA + 0.1 mg kinetin + 1.0 mg GA3/litre gave the highest rate (80%) of plantlet formation.

10 Kokubu et al (1993) studied the Ipomoea batates callus which induced from shoot tip explants on media containing various concentrations of growth regulators; best results were with 0.5 mg kinetin and 0.02 mg 2, 4-D /litre. When calluses were transferred to regeneration media containing 0 or 0.2 mg IAA and 0.0, 1.0 or 2.0 mg benezyladenine (BA)/litre after 8 weeks, only those calluses which were induced on media containing 0.02 mg 2, 4-D and 0 or 0.5 mg kinetin/litre formed roots. Six weeks later, rooted calluses were transferred to fresh regeneration medium and then onto basal medium. Five weeks later shoots formed from petiole-derived calluses which had been cultured on the callus-induction medium containing 0.02 mg 2, 4-D and 0.5 mg kinetin /litre and then on the regeneration medium containing 0.2 mg IAA and 1.0 mg BA/litre. Plantlets were obtained after 4 weeks. Pido and Kowyama, (1995) found Ipomoea trifida that the shoot meristems of 8 lines were cultured on C1 medium (MS medium containing 2.21 mg 2, 4-D) and the highest frequency of embryogenic callus formation (50%) was obtained from H104-28 line. The frequency of embryogenic callus formation on shoot meristems of H104-28 when cultured on C1 and C2 medium (MS medium without vitamins and 2.21 mg 2, 4-D/litre) was 40 and 56, respectively. Plant regeneration from embryogenic calluses of H104-28 was achieved either through immediate transfer of calluses to regeneration medium (MS medium supplemented with 0.2 mg NAA/litre) or through transfer to somatic embryo production medium (MS medium supplemented with 2.64 mg Abscisic acid/litre) followed by regeneration medium. Immediate transfer of calluses to regeneration medium resulted in a higher rate of plant regeneration. Shahnewaz Sharmin and Bari (2004) studied the effect of various concentrations of sucrose in culture media on the frequency of callus induction and regeneration of green plantlets from anther culture of rice were investigated. Cold pretreated anthers of BRRI Dhan-29 at 6_C for seven days were cultured on N6 medium containing sucrose at concentrations of 0, 1, 2, 3,

11 4, 5, 6% ; 2.0 mg/l 2,4-D ; 0.5 mg/l Kin and 2.5 mg/l NAA. Results revealed that 4% sucrose was suitable for inducing high frequency (7.5%) callus induction and high green plant regeneration (65%). Highest concentration of sucrose (6% and above) in the culture medium not only resulted in an increase in the percentage of callus formation but also prompted the regeneration of albino plants. Regeneration from male gametes has been reported in more than 200 species belonging to the Solanaceae, Cruciferae and Gramineae families while many legumes and woody plants are rather recalcitrant Raghavan 1990; Wenzel et al. 1995 and Germana, 2009). Embryogenesis in pollen is normally induced through anther or isolated microspore culture. Anther culture is often the method of choice for DH production in many crops because the simplicity of the approach allows largescale anther culture establishment and application to a wide range of genotypes (Sopory and Munshi, 1996). The technique of isolated microspore culture, performed by removing somatic anther tissue, requires better equipment and more skills compared to anther culture, although the former provides the better method for investigating cellular, physiological, biochemical and molecular processes involved in pollen embryogenesis Nitsch (1977) introduced the concept of ‘‘Wall Factor’’, according to which the somatic tissues of the anther play an important role in the induction of sporophytic divisions in pollen, with the diffusion of nutrients through the anther walls often considered to be one of the factors affecting microspore embryogenesis. A number of studies on the role of the anther wall in pollen embryogenesis have shown that it not only acts as a barrier to nutrient flow but that it also provides both beneficial and inhibitory substances Pulido et al. (2005) using both culture systems (anther and isolated microspore culture) to induce microspore embryogenesis in barley, showed that the initial phases of both processes are similar. These researchers as well demonstrated that the anther wall also served as a filter by preventing excessive concentrations of Fe around the microspores within the anther, even when the concentrations

12 present in the culture medium were high. The first natural sporophytic haploid was observed in 1921 by Bergner in a weed species Datura stramonium L. and reported by (Blakeslee et al., 1922). The importance of haploids in plant breeding and genetic research was immediately recognized. The number of spontaneous haploids detected has steadily grown, and Kasha (1974) recorded the occurrence of over 100 angiosperm species. A list of selected examples of occasional haploids in a range of species has been reported by (Dunwell, 2010). The frequency of spontaneous haploids is, however, too low for practical application in breeding. About 40 years after the identification of the first natural haploid, Guha and Maheshwari (1964) discovered that it was possible, by in vitro culture of immature anthers of the Solanaceous specie Datura innoxia, to change the normal gametophytic development of microspores into a sporophytic one and that embryos and plants with a haploid chromosome number would then be produced. This discovery gaved the way to further and extensive research on anther culture that was particularly successfully in the Solanaceae, Brassicaceae and Gramineae. However, not all of the angiosperm crops of interest efficiently respond to embryogenesis induction, and although barley (Hordeum vulgare L.), rapeseed (Brassica napus L.), tobacco (Nicotiana spp.) and wheat (Triticum aestivum L.) are considered to be model species to study microspore embryogenesis due to their high regeneration efficiency (Forster et al., 2007), other scientifically or economically interesting species, such as Arabidopsis, many woody plants or members of legume family, still remain recalcitrant to this type of in vitro morphogenesis (Raghavan, 1990; Wenzel et al. 1995 and Germana, 2009). Gamete embryogenesis is a particularly indispensable tool for obtaining homozygosity in woody plants, which are characterized by a high genome heterozygosity, a long generation cycle with a long juvenile period, a large size and, often, self-incompatibility, and for which it is not possible to obtain haploidization through conventional methods, i.e., several generations of selfing (Germana, 2009). The great interest in

13 haploids was appearent with the organization of the First International Symposium ‘Haploids in Higher Plants’, which took place at Guelph (Canada) in 1974 (Kasha, 1974). In the early 1970s, cv. Maris Haplona of rapeseed (Brassica napus) was the first DH crop plant released (Thompson, 1972), followed by cv. cultivar Mingo in barley (Hordeum vulgare) in 1980 (Ho and Jones, 1980). Since then, a great deal of research has been carried out with the aim of establishing efficient techniques for haploid and DH production with an increasing number of genotypes. For a long time, many postulations regarding pollen embryogenesis protocols have been based on practical experience. However, recent scientific and technological innovations, a greater understanding of underlying control mechanisms and an expansion of end-user applications have induced a resurgence of interest in haploids in higher plants (Forster et al., 2007). This interest is shown by the establishment of the COST 851 programme, a European Union-funded research network entitled ‘Gametic cells and molecular breeding for crop improvement’, that ran from 2001 to 2006. To date, almost 300 new superior varieties belonging to several families of the plant kingdom (particularly annual crops) have been produced. A variety of methods were used to obtain these DHs, such as chromosome elimination subsequent to wide hybridization, the ‘‘bulbosum’’ method by (Kasha and Kao, 1970), pollination with irradiated pollen, selection of twin seedlings, in vivo or in vitro pollination with pollen from a triploid plant, gynogenesis and pollen embryogenesis through in vitro anther or isolated microspore culture (Forster and Thomas, 2005). The website provides a list of haploid-derived varieties, mostly Asparagus, Barley, Brassica, Eggplant, Melon, Pepper, Rapeseed, Rice, Swede, Tobacco, Triticale and Wheat. The application of intellectual property (IP) protection and the patenting system of haploid plants (where patents also include anther and microspore culture techniques) has been reviewed by Dunwell (2010), who added that the strong commercial interest in methods for the production and exploitation of haploid plants is exemplified by

14 the extensive number of granted patents and patent applications from the USA and elsewhere.

Diplodization. Many vegetatively propagated flowers and fruits as well as cultivated agricultural crops are polyploids. Since natural polyploidy does not exist in all genera and because of the interesting effects obtained by polyploidization, polyploids of several economically important crops have been artificially induced over the past decades. The first applications of mitotic polyploidization in agriculture were reported in the 1930s (Blakeslee and Avery, 1937). This initial polyploidization was induced on plants established in soil. Colchicine was the most commonly used antimitotic agent. Roots were soaked in or mea- nwhile axillary buds were wetted with a colchicine solution (Pei, 1985). Blakeslee (1939) reported the successful production of polyploids in 48 different species using colchicine; for agricultural crops, in vivo chromosome doubling was successfully applied for Sugar and Fodder beet, Ryegrass and Red clover (Dewey, 1980). Despite the effectiveness of the method, low rates of polyploidized plants were frequent and high numbers of mixoploids were retrieved (Pei 1985).

Colchicine is the most commonly used agent to induce chromosome doubling (Kihara, 1951). The Colchicine has been used to double the chromosomes in Watermelon but it is moderately toxic to human & animals and expensive (Ying et al., 1999). Baenziger and Schaeffer (1983) indicated that anthers give plant breeders a view of how dihaploids could be used in crop improvement. Anther culture techniques, dihaploid breeding theory, present uses of dihaploids in breeding and future use of dihaploids in plant breeding (mainly in the development of new cultivars, in biochemical selection breeding and gene transfer systems). Burun and Emiroglu (2008). In order to obtain androgenic doubled haploids of tobacco (Nicotiana tabacum cv.

15 Karabaglar 6265), colchicine was applied at 3 different stages of anther culture. Before culture, the anthers were treated with 0.4% aqueous solution of colchicine for 0, 2, 4, 6, 8, 10 and 12 h. Culture response of anthers decreased as the treatment duration increased (except for 12 h) and the highest diploidization of 29.7% was obtained with 6 h. During culture, when macroscopic embryoids appeared in dehisced anthers, they were wrapped in sterile cotton saturated with 0.2% colchicine for 3 days and transferred to fresh medium. This application resulted in 60% diploidization; however, plant recovery from the treated embryoids was low and only 5 plants could survive. When plantlets with 4 to 8 leaves immersed in 0.2% colchicine for 0, 7, 24 and 48 h on a shaker, a side from 4.3, 42.3, 37.8 and 33.3% doubled haploids, respectively, haploids, tetraploids, aneuploids, and mixedploids were also found among the treated plants. The main advantage of this method is that treated plantlets can be transplanted directly into pots to grow androgenic plants. When chromosome doubling rate and viability are taken into consideration, among the 3 methods tested, plantlet treatment with 0.2% colchicine for 7 h appeared to be more efficient with 42.3% dihaploids. Durations shorter than 7 h must be tested to optimize the application.

Radiation effect on haploid plant induction Gamma rays are the most energetic form of such electromagnetic radiation as X- rays, visible light and UV, having the energy level from around 10 kiloelectron volts to several hundred kiloelectron volts. Gamma rays more penetrating than other radiation as alpha and beta rays (Kovács and Keresztes, 2002). The principle site of the damage caused by ionizing radiation to plants is in the cell nucleus (Sparrow & Woodwell, 1962). Radiobiological studies on plant tissues in culture may provide information on the cell growth behavior, radiosensitivty and the induction of mutations. The radio sensitivity of plants and calli can be manifested mostly in three ways; 1) inhibition of growth, 2) reduction of reproduction capacity and 3) Death. Growth inhibition induced by ionizing

16 radiation has been attributed to chromosome deletion (Sparrow et al., 1961) and changes in a variety of biochemical and physiological systems (Gunckel and Sparrow, 1961). In general, the effects of gamma irradiation on any material have been summarized by (Willard, 1955) who emphasized that all radiations loose their energy on passing through a material producing electronically excited or ionized molecules. The chemical effects follow as a result of the transfer of energy of excited electron (in ionized molecule) into vibrational energy sufficient to break bonds in the molecule. Most of these chemical events results in the formation of free radicals which may combine with each other or react with other species of the medium and form a different molecules.

The effect of ionizing radiation induces many hinds of injuries and damage, which affect growth, metabolism and differentiation. Since this radiation is highly penetrating the resistance against it is mainly based on toleration mechanisms (Levitt, 1980). The use of mutagenesis in vitro induce mutation showed provide a new input to the solution of breeding problems in crop plants (Ancora and Sonnino, 1987). Balite et al. (1989) cited that the haploid and diploid calli of Nicotiana glauca (n=12) and N.langsdorffii (n=9) together with diploid calli of N. tabacum (n=24) cv. Bright Yellow which were taken as control were exposed to various dose of 60Co gamma rays at 7 days and 10 days after incubation.Calli were subsequently weighted for a period of 12-14 days after radiation (19-24 days after incubation) and the effect of irradiation on the growth was studied. A sigmoid curve observed for the growth rate as well as for the growth pattern. The radio sensitivity were estimated on the basis of the following different two criteria; The difference in the diploid callus volumes at 12 or14 days after irradiation and at the time of radiation .2) The growth rate of diploid and haploid calli during these period. In 2 species except in N . langsdorffii, calli irradiated ten dayse after incubation (at the beginning of the actively growing stage) were more sensitive to gamma irradiation than calli irradiated seven

17 days after incubation (at a relatively slow growing stage). Each species had its own distinctive color before irradiation; golden yellow, green and yellow green for Nicotiana glauca, N.langsdorffii and N. tabacum; respectively. The color of some of the calli tended to darken with increasing irradiation dose.When the rate of increase of the irradiated calli to that of the unirradiated ones was used as an indicator of radio sensitivity, there was a significant difference in the radio sensitivity of the calli among the species: N. tabacum was more sensitive followed by Nicotiana glauca and N.langsdorffii, which was the least sensitive. On the other hand, when the curve of the increase to the growth rate was used as the criterion of radio sensitivity, N.langsdorffii appeared to be more sensitive than N. tabacum and Nicotiana glauca. Haploid calli were more radio sensitive than diploid ones in Nicotiana glauca, but no significant difference was observed between them in N.langsdorffii. Hasegawa et al (1995) studied that the tobacco (Nicotiana tabacum ) anthers which had been cultured for (0, 10 and 20 days) were irradiated with gamma radiation doses (0, 2.5, 5, 10, 20, 40, 80 and 160 Gy). Anthers cultured for 10 days before irradiation, which were correspondence to a plantlet initiation stage of microspore, were most radiosensitive and RD50 for relative anther culture response (RACR) (defined as dose resulting in 50% reduction in) was 8 Gy. On the other hand, RD50s for RACR in the anthers precultured for 0 and 20 days were 50 Gy. The highest frequency of morphological variants was also obtained from the plants derived from irradiated anthers precultured for 10 days. The gamma irradiation on the anthers precultured for 10 days is most effective in inducing mutation in tobacco. Golz et al (1999) stated that the mutations affecting the self-incompatibility response of Nicotiana alata were generated by irradiation with 8 Gy of gamma radiation. Mutants in the M1 generation were selected on the basis of pollen tube growth through an otherwise incompatible pistil. Twelve of the 18 M1 plants obtained from the mutagenesis screen were self-

18 compatible. Eleven self-compatible plants had mutations affecting only the pollen function of the S locus (pollen-part mutants). The remaining self-compatible plant had a mutation affecting only the style function of the S locus (style-part mutant). Cytological examination of the pollen-part mutant plants revealed that 8 had an extra chromosome (2n + 1) and 3 did not. The pollen-part mutation in 7 M1 plants was followed in a series of crosses. DNA blot analysis using probes for S-RNase genes (encoding the style function of the S locus) indicated that the pollen-part mutation was associated with an extra S allele in 4 M1 plants. In 3 of these plants, the extra S allele was located on the additional chromosome. There was no evidence of an extra S allele in the 3 remaining M1 plants. The breakdown of self-incompatibility in plants with an extra S allele is discussed with reference to current models of the molecular basis of self- incompatibility.

Mkuya et al (2005) suggested that the panicles of an Indica rice line TM7-5 were subjected to radiation with 137Cs gamma rays at 0 (control), 5, 10, 15 and 20 Gy; respectively, and then its anthers were cultured. There were slight differences among the treatments in peak emerging time of callus initiation, from 38 to 44 days after inoculation (DAI) as well as the frequency of callus initiation (2.3-3.5%). About two thirds calli were induced before 44 DAI, and calli derived beyond 60 DAI lost the regeneration ability. Green plant regeneration frequency was significantly stimulated from two- or three-fold by irradiation of the 137Cs gamma rays compared with the control, and the maximum was 22.81% (15 Gy). The culture ability based on callus initiation and green plantlet regeneration was 0.19% for the control while it was over 0.45% for all the irradiated treatments, and the maximum was 0.59% for 15 Gy treatments. Ionizing radiation is known to have several effects on plant growth and development, ranging from stimulatory effects at very low doses, harmful consequences at intermediate levels and pronounced detrimental outcomes at high doses. The severity of

19 the effects is dependent upon several factors including species, cultivars, plant age, physiology and morphology as well as plant genome organization (Holst and Nagel, 1997). The different response of varieties and ecotypes within the same species can be ascribed to their different radiosensitivity due to different levels of ploidy or controlled by different genetic organization such as polygenes and single major gene systems (Ukai and Yamashita, 1969 and Al-Rubeai and Godward 1981). It is well known that the resistance of plant seeds is stronger than that of animal cells (Casarett 1968; Kumagai et al., 2000 and Real et al., 2004). Indeed, the seed is a peculiar stage of plant life cycle, characterized by higher resistance to environmental factors, because of its structural and metabolic traits, when compared with organs at different life stages. There are also differences in sensitiveness to irradiation between dry and fresh seeds because of their water content and structures that affect the capacity of the ions to penetrate and reach the embryo (Yu, 2000; Wu and Yu, 2001 and Qin et al. 2007). Complex tissue organization seems to be more resistant to the harmful and mutagenic effects of radiations because the multicellular status allows cell and tissue repair (Shikazono et al. 2002 and Durante & Cucinotta, 2008). The first attempts to stimulate plant growth by exposing seeds or growing plants to low doses of ionizing radiation or by the use of radioactive fertilizers, date back to the sixties (Sax,1963). Low- and high-LET (Linear Energy Transfer) ionizing radiations have been widely used in breeding programs because they are expected to cause mutations with a wide spectrum Zhou et al. (2006).

For the molecular nature of heavy ion–induced mutations in plants, for their transmissibility and for a description of the novel phenotypic characteristics observed after heavy-ion exposure on ground, we refer to a recent review (Tanaka et al., 2010). The reason why heavy ions are more suitable for induced mutagenesis is well explained by Shikazono and co-workers (Shikazono et al. 2002 and 2003) as reported in Tanaka et al. (2010). In addition to this study another interesting tool to

20 investigate genomic stability in plants is represented by the recombination assay as shown by Kovalchuk et al. (2000). The same author used transgenic seeds and seedlings of Nicotiana tabacum and Arabidopsis thaliana, with a non-functional version of the b-glucoronidase marker gene, demonstrated that events of homologous rejoining (HR) can restore the genetic function. After exposure to acute or chronic c-rays, they observed an increased frequency of HR in both species, particularly in A. thaliana. The effect was more pronounced when seedlings were irradiated, due to more active metabolism and higher water content. Interestingly, the pattern of recombination events was quite homogenously distributed in leaves, stem and root when seeds were irradiated, while a prevalence of HR events in leaves was observed irradiating 10- day-old plants, suggesting a higher sensitivity of these sectors of the plant. Comparing high- and low-dose-rate exposures, the authors found that chronically irradiated samples show a higher genomic instability compared to acutely exposed samples, even though the absorbed doses where several orders of magnitude lower. The exposure of plants to ionizing radiations determines changes of the genetic material that are responsible for the alteration in mitotic and meiotic capacity of cells, thus resulting in modification of growth and reproductive processes. In plants, also totipotency, hence morphological differentiation, is affected by radiation exposure. On the other hand, radiations have an important role in the induction of mutations that are revealed through various changes in the phenotype. Many studies have been carried out to evidence the effects of high radiation levels on the mitotic/meiotic ability, totipotency and differentiation of plant cells, through the analysis of various parameters such as germination and survival rate, lethality, organ differentiation, tumourization, occurrence of morphological aberrations, flowering rate and sterility (Wu and Yu, 2001; Durante and Kronenberg, 2005 and Zhou et al., 2006). As for mutational analysis, available results are not always easily comparable mainly because of the utilization of different methodologies during irradiation (different radiation type, doses and exposure

21 times) and of the use of several parameters to describe plant growth with ambiguous definitions. Lethality is often used as the opposite of survival that is defined in various ways. More specifically, survival is described either as the percentage of plants with a minimum number of expanded leaves after an established number of days from sowing or as the number of plants that are able to reach the reproductive phase and can produce seeds (Kumagai et al. 2000, Wu and Yu, 2001 and Shikazono et al., 2002). Whatever the definition lethality and survival are much more complex concepts than germinability because they involve the further growth of viable plants with true leaves (Wareing and Phillips, 1981). The interpretation of results and the identification of common behaviours are complicated by the variability of reactions found in different species and cultivars (Bayonove et al., 1984 and Yu et al., 2007). Also polyploidy reduces the radiosensitivity of plants; for example, there is evidence that, after high- LET irradiation, tetraploid lines of A. thaliana survive longer than diploid lines that are more easily subjected to the lack of germination or early death of seedlings (Gartenbach et al., 1996). As we explained in the previous section, polyploidy can hide recessive mutations (including the ones affecting the lifespan of the plant) due to the existence of supernumerary copies of the genome.

Adaptation

Wardle et al (1983) found that the acclimatization can take place by allowing the in vitro plants to gradually get used to a lower relative humidity, which is the case in vivo. Development of stomata closure mechanism is a very important component acclimatization, have been showed with Brasica oleracea Botrytis that lowering of the relative humidity in vitro results in better wax formation on the cuticular evaporation formation on the cuticular layer, leading to less cuticular evaporation . Sutter and Hutzell (1984) observed that acclimatization directly after the in vitro phase can be brought about by keeping

22 the relative humidity high in vivo and maintaining a low irradiance and temperature. Another method of acclimatization is to leave the tube or the flask open in a sterile environment for a few days to adjust to in vivo conditions. It is possible to spray the plants with anti-transpirants to reduce evaporation in vivo, although this often has an adverse effect. Pierik (1987) cited that the plant which has originated in vitro, differs in many respects from one produced in vivo.The plants grown in test tubes their cuticle (wax layer) is often poorly developed, because the relative humidity if often 90- 100% in vitro.This results in extra water loss through cuticular evaporation, when the plant is transferred to soil, since the humidity of the air in vivo is much lower leaves of in vitro plants, often thin, soft, and photosynthetically not very active are not well adapted for the in vivo climate .Test tube plants have smaller and fewer palisade cells to use light effectively, and have larger mesophyll air space. Stomata don’t operate properly in tissue culture plants, open stomata in tissue culture plants cause the most significant water stress during the first few hours of acclimatization. In tissue culture plants poor vascular connection between the shoots and roots may reduce water conduction. It most also be realized that the in vitro plant has been raised as a heterotrophy, while it must be autotrophic in vivo sugar must be replaced through photosynthesis . Ziv (1992) found that the roots that have originated in vitro appear to be vulnerable and not to function properly in vivo (few or no root hairs ).They quickly die off and must be replaced by newly formed subterranean roots. The development of root hairs in vitro can sometimes be promoted by allowing them to develop in a liquid medium . The poorly developed root system makes in vivo growth for such a plant very difficult, especially when there is high evaporation. It is vital that the in vitro plant looses as little water as possible in vivo. Acclimatization in vitro especially by exposing the plants to reduced relative humidity increases the survival rate when the plants are transferred to soil.

23 Carneiro et al (1997) illustrated that S. rebaudiana contains sweeteners, which are sweeter than sucrose. The effects of 14 growing media on greenhouse seedling production of S. rebaudiana were investigated in Brazil. The best mixture for seedling growth consisted of sand clay loam soil, hen manure (10% v/v) and lime. Nepovim and Vanek (1998) reported that the culture of multiple shoot culture of Stevia rebaudiana in the bioreactor and transfer to ex vitro conditions took about 8-9 weeks. A total of 600 plantlets were produced which could be transferred from greenhouse to field conditions. Metry et al (2003) found that the different combinations of peatmos and sand for adaptation of Stevia rebaudiana plantlets derived through micro propagation method. The survival percentage ranged from 20-80 % , the best potting medium was sand –peat at 50%(v/v).

Haploid plants under Salinity stress Marla et al. (1985) found that the cell lines of tobacco (Nicotiana tabacum L. var Wisconsin 38) were obtained which are adapted to grow in media with varying concentrations of NaCl, up to 35 grams per liter (599 millimolar). Salt-adapted cells exhibited enhanced abilities to gain both fresh and dry weight in the presence of NaCl compared to cells which were growing in medium without NaCl (unadapted cells). Tolerance of unadapted cells and cells adapted to 10 grams per liter NaCl was influenced by the stage of growth, with the highest degree of tolerance exhibited by cells in the exponential phase. Cell osmotic potential and turgor varied through the growth cycle of unadapted cells and cells at all levels of adaptation, with maximum turgor occurring at approximately the onset of exponential fresh weight accumulation. Adaptation to NaCl led to reduced cell expansion and fresh weight gain, while dry weight gain remained unaffected. This reduction in cell expansion was not due to failure of the cells to maintain turgor since cells adapted to NaCl underwent osmotic adjustment in excess of the change in water potential caused by the addition of

24 NaCl to the medium. Tolerance of the adapted cells, as indicated by fresh or dry weight gai, did not increase proportionately with the increase in turgor. Adaptation of these glycophytic cells to NaCl appears to involve mechanims which result in an altered relationship between turgor and cell expansion. Salinity stress biology and plant responses to high salinity have been discussed over two decades (Ehret and Plant, 1999; Hasegawa et al., 2000 and Zhu, 2002) and it has been over a decade since salinity tolerance in marine algae has been covered (Kirst, 1989). These reviews covered organismal, physiological, and the then-known biochemical hallmarks of stress and the bewildering complexity of plant stress responses. We summarize in this review physiological, biochemical, and molecular mechanisms of salt tolerance with the salient features of salinity stress effects on plants. In this review, much research information about cellular, metabolic, molecular, and genetic processes associated with the response to salt stress, some of which presumably function to mediate salt tolerance, has been gathered. Parida, and Das, (2005) exposed plants to salt stress undergo changes in their environment. The ability of plants to tolerate salt is determined by multiple biochemical pathways that facilitate retention and/or acquisition of water, protect chloroplast functions, and maintain ion homeostasis. Essential pathways include those that lead to synthesis of osmotically active metabolites, specific proteins, and certain free radical scavenging enzymes that control ion and water flux and support scavenging of oxygen radicals or chaperones. The ability of plants to detoxify radicals under conditions of salt stress is probably the most critical requirement. Many salt-tolerant species accumulate methylated metabolites, which play crucial dual roles as osmoprotectants and as radical scavengers. Their synthesis is correlated with stress-induced enhancement of photorespiration. In this paper, plant responses to salinity stress are reviewed with emphasis on physiological, biochemical, and molecular mechanisms of salt tolerance. This review may help

25 in interdisciplinary studies to assess the ecological significance of salt stress. Ahmad (2008) determined that the salinity limits of the production capabilities of agricultural soils in large areas of the world. Both breeding and screening germplasm for salt tolerance encounter the following limitations: (a) different phenotypic responses of plants at different growth stages, (b) different physiological mechanisms, (c) complicated genotype × environment interactions, and (d) variability of the salt-affected field in its chemical and physical soil composition. Plant molecular and physiological traits provide the bases for efficient germplasm screening procedures through traditional breeding, molecular breeding, and transgenic approaches. However, the quantitative nature of salinity stress tolerance and the problems associated with developing appropriate and replicable testing environments make it difficult to distinguish salt-tolerant lines from sensitive lines. In order to develop more efficient screening procedures for germplasm evaluation and improvement of salt tolerance, implementation of a rapid and reliable screening procedure is essential. Field selection for salinity tolerance is a laborious task; therefore, plant breeders are seeking reliable ways to assess the salt tolerance of plant germplasm. Salt tolerance in several plant species may operate at the cellular level, and glycophytes are believed to have special cellular mechanisms for salt tolerance. Ion exclusion, ion sequestration, osmotic adjustment, macromolecule protection, and membrane transport system adaptation to saline environments are important strategies that may confer salt tolerance to plants. Cell and tissue culture techniques have been used to obtain salt tolerant plants employing two in vitro culture approaches. The first approach is selection of mutant cell lines from cultured cells and plant regeneration from such cells (somaclones). In vitro screening of plant germplasm for salt tolerance is the second approach, and a successful employment of this method in durum wheat is presented here. Doubled haploid lines derived from pollen culture of F1 hybrids of salt-tolerant parents are promising tools to further improve salt tolerance of plant cultivars. Enhancement

26 of resistance against both hyper-osmotic stress and ion toxicity may also be achieved via molecular breeding of salt-tolerant plants using either molecular markers or genetic engineering.

Nazar et al., (2011) taught that the salinity is a major abiotic stress factor affecting plant growth and productivity worldwide. The salinity induced reduction in photosynthesis, growth and development of plants is associated with ionic/osmotic effects, nutritional imbalance or oxidative stress. Plants develop several mechanisms to induce tolerance to overcome salinity effects. Of the several possible mechanisms to reduce the effects of salinity stress, management of mineral nutrients status of plants can be the efficient defense system. Sulfur (S) is an important plant nutrient involved in plant growth and development. It is considered fourth in importance after nitrogen, phosphorus, and potassium. It is an integral part of several important compounds, such as vitamins, co-enzymes, phytohormones and reduced sulfur compounds that decipher growth and vigor of plants under optimal and stress conditions. The present review focuses on improving our understanding on the salinity effects on physiology and metabolism of plants and the importance of sulfur in salinity tolerance.

Chemical Analysis Proline and Protein contents

As known, proteins are the end-products of transcription and translation of the genetic information. They fulfill many important evolutionary levels, genetic expression and natural selection (protein is part of the phenotype), they contain very valuable information to help understanding phylogeny and (Jensen and Fairbrothers 1983).

Sabry et al (1995) tested six cultivars of spring wheat for osmoregulatio under drought stress (imposed by withholding irrigation for three days) or salinity stress (imposed by irrigation with 200 mM NaCl for 10 days). They found that wheat

27 seedlings accumulated sucrose, glycine betaine, proline, asparagine, glutamine and valine. Interestingly, proline was the predominant osmolyte observed in drought stressed plants, but glycine betaine and sucrose were the leading osmolytes in salt- stressed plants. Significant variation was observed at the levels of many of these osmolytes in these cultivars. They concluded that this observation might be useful in the development of drought and salinity tolerant cultivars.

Agastian et al. (2000) reported that soluble proteins increased at low salinity concentration and decreased at high salinity concentration in mulberry. On the other hand, soluble protein of cow pea plants slightly reduced by salt treatments during the short- term exposure, while the plants pre-acclimated with NaCl exhibited lower concentrations of proteins and amino acids when compared with non-acclimated plants (Silveira et al., 2001). In line with this finding, biochemical studies showed the accumulation of amino acids and proteins are regarded as a common response of plants such as barley and wheat (El- Shintinawy and El- Fiky, 2001) to salinity stress.

Abo El-Seoud et al., (1991) stated that the individual amino acids of leaves of Atropa belladonna exhibited a diversity in their response to the applied irradiation doses (80, 160, 240 and 320 Gy) of gamma rays, where there was a slight increase or decrease in their content. In general total amino acid in all doses increased slightly. Other abiotic stresses,can induce oxidative stress through the increase in relative oxygen species (ROS), which may cause cellular damage through oxidation of lipids, proteins and nucleic acids (Demiral & Turkan, 2005 and Khan & Panda, 2008). The changes in DNA, RNA and protein contents either increase or decrease may have the potentiality to alter the transition process or the transcriptional one via affecting and or modulating the activity of some enzymes responsible for enhancement of protein synthesizing machinery, (Abd El- Hamid, 2000 , Shehab et al., 2004 and Morsi, 2008).

28 In response to severe changes in environmental conditions, higher plants are able selectively to activate or inhibit the expression of specific genes, leading to the accumulation of particular sets of proteins, which are referred to as stress – induced proteins (Scandalias & Baum 1982) and Ho & Sachs,1989). A large number of stress induced proteins have been discovered in cases of heat shock, salt stress, wounding, anaerobiosis, microbial infection, etc. For most of these proteins, very little is known about their cellular role and the molecular mechanisms involved in the induction of their synthesis (signal transduction). It has been suggested that oxidative signals may play a control role in the cellular response to stress. In plant, the activation of molecular oxygen, leading to the formation of oxygen centered free radical, has been observed in tissues exposed to different stress conditions, such as UV, certain herbicide and plant microorganism interactions (Elstner et al., 1988) and Sutherland, 1991). (Ferullo et al., 1994) found that the processes of protein synthesis do not seem to be qualitatively affected. Although DNA impairment might be expected after massive irradiation, the appearance of the new sets of translation products and proteins can not be attributed to a dysfunction of transcription and translation; the changes in gene expression concern very specific products and are probably caused by a regulated modification of gene expression, in response to a specific stress situation. As a result, gamma- induced proteins could be considered as a particular class of stress- induced proteins. Although it would be difficult to pinpoint any direct relationship between gamma- induced proteins and the different translation products by irradiation, the appearance of new translatable RNAs indicates that at least a part of this stress response occurs at the transcriptional level. Several new protein which are synthesized in response to an altered environment have reported as “stress proteins” as shock proteins in plants (Ericson and Al – Finito1984, Kanabus et al., 1984 and Oliver and Bewley 1984). However, only a few of these proteins have been found to be involved in known physiological or metabolic processes (Hanson &Jacobson,

29 1984 and Hasegawa et al. 1984). Most of these proteins appear as immediate response by the organism to an altered environment (temperature, osmotic stress and wounding) and it is questionable whether many of them are associated with increased growth and survival of the plants in new environment.

Tanaka et al. (1992) found that the 10 proteins were induced and about 15 proteins were reduced after gamma irradiation treatments in Deinococcus radiodurans.

Tuna et al. (2008) showed that the plants grown at 100 mM NaCl produced less dry matter and chlorophyll content than those without NaCl. Proline content and sodium (Na)+ concentration in the leaves increased by the addition of 100 mM NaCl. The accumulation of Na in roots indicates a possible mechanism whereby bread wheat copes with salinity in the rooting medium and/or may indicate the existence of an inhibition mechanism of Na+ transport to leaves. Concentrations of both Ca2+ and K+ were lower in the plants grown at high NaCl.

Molecular Markers in N.alata; many others were revealed about the genetical markers in N.spp. by using SDS – PAGE and DNA RAPD.

1- SDS-PAGE electrophoresis

Mary and Sharon (1984) Found that the protein pattern of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that have become adapted to a medium containing 10 grams NaCl per liter was compared to that of unadapted cells on one dimensional sodium dodecyl sulfate gels. Two protein bands (32,000 and 20,000 KDa) were much more abundant in the salt adapted cells, and one protein (26,000 KDa) was unique to the salt cells. This protein pattern did not change during the growth cycle of the cells. When salt adapted cells are transferred to control medium, their ability to grow in the salt-containing medium returns to that of control cells after one passage in the

30 control medium within this time the levels of the 32,000 and 20,000 KDa proteins also return to that of the control cells, but the 26,000 KDa protein does not disappear until after at least two passages in control medium. Amino acid analyses of these three proteins revealed that they all contain some hydroxyproline. Narendra et al. (1985) cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands are reduced. Enhanced levels of 43- and 26- KDa polypeptides are present in both NaCl and PEG- induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced and reduced levels of polypeptide bands. Synthesis of 26- KDa polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate 3sS into a 26- KDa polypeptide during the later stage of culture growth beginning at midlog phase. The 26- KDa polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunological cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26- KDa polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. Capkova et al (1988) studied that the tobacco cv. Samsun, , N. alata and apple pollen tubes grown in vitro were pulse-labelled for 5-30 min with [14C] amino acids, and the proteins analysed by SDS-PAGE and fluorography. In tobacco, the qualitative pattern of the synthesized proteins did not change significantly when the pollen was cultured for 1-24 h in different media, but total protein synthesis decreased as pollen tube growth slowed down and was very low in non-growing tubes. The patterns showed

31 preferential synthesis of proteins with apparent MW of 63 and 65 kDa. The 65 kDa species, which was not synthesized during pollen maturation and was absent in ungerminated pollen grains, appeared as the most intensely labelled band in both soluble and insoluble protein fractions, but accumulated as a protein non- covalently bound to pollen structures. It was also preferentially synthesized in N. sylvestris, N. alata and apple pollen tubes, and probably plays an important role in tube wall formation. In tobacco, the labelling of the 65 kDa protein was not specifically reduced upon strong inhibition of transcription by actinomycin D; this indicates a post-transcriptional level of induction of its synthesis in germinating pollen. Ashraf and Harris (2004) despite a wealth of published research on salinity tolerance of plants, neither the metabolic sites at which salt stress damages plants nor the adaptive mechanisms utilized by plants to survive under saline conditions are well understood. As a result, there are no well-defined indicators for salinity tolerance available to assist plant breeders in the improvement of salinity tolerance of important agricultural crops. Although plant breeders have successfully improved salinity tolerance of some crops in recent decades, using plant vigor or seed yield as the main selection criteria, selection may be more convenient and practicable if the crop possesses distinctive indicators of salt tolerance at the whole plant, tissue or cellular level. Thus, there is a need to determine the underlying biochemical mechanisms of salinity tolerance so as to provide plant breeders with appropriate indicators. the possibility of using these biochemical characteristics as selection criteria for salt tolerance is discussed. It is concluded that although there are a number of promising selection criteria, the complex physiology of salt tolerance and the variation between species make it difficult to identify single criteria. Progress is more likely if biochemical indicators for individual species rather than generic indicators can be determined. Anna et al (2008) reported that in vitro mutagenesis by means of gamma irradiation can be used to produce economically improved mutants. Gamma irradiation was used to

32 induce biochemical changes in a prominent medicinal plant,Orthosiphon stamineus. Shoot tip explants of O. stamineus were subjected to gamma irradiation. Biochemical studies on O. stamineus revealed that the total soluble protein and total chlorophyll content decreased notably as the gamma dosage increases. However, plantlets irradiated at 10 and 20 Gy exhibited total soluble protein content were higher than all other treatments. Severe increased in specific activity of peroxidase was observed especially in plantlets irradiated at high dosages (70Gy). Analysis on protein banding profile of O.stamineus demonstrated that gamma irradiation at the treatment dosages did not produce a significant change in protein constituent. Nine major bands of relatively similar intensities were observed. Rosmarinic acid content was the in plantlets irradiated at 30 Gy. However, it was suggested that gamma irradiation at low dosages did not produce an apparent change in rosmarinic acid content as an irregular trend of production profile was observed. Due to the potential future of O. stamineus in pharmaceutical industries, further research on this plant could be done to facilitate the propagation of superior mutants that is able to produce vast quantities of valuable secondary metabolites.

2-Randomly amplified polymorphic DNA (RAPD)

Bai at al (1995) studied the linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of 'Delgold' tobacco, a BCsFs near isogenic line (NIL) carrying the resistance gene in a 'Delgold' background, and 'PB19', the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between 'Delgold' and 'PB19', indicating there are few RAPD polymorphisms between them despite relatively

33 dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between 'Delgold' and the NIL. All of these markers proved to be unlinked with the resistance gene in F 2 linkage tests. Of the remaining three RAPD markers polymorphic between 'Delgold' and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene .

Filippis et al (1996) studied the positive identification of somatic hybrids generated through protoplast fusion is often difficult or impossible based primarily on morphological or biochemical analysis. The use of molecular markers potentially offers a reliable and repeatable technique, allowing early assessment of polymorphic variation of hybrids directly at the DNA level. The random amplification of polymorphic DNA (RAPD) by polymerase chain reaction (PCR) to analyse the relationship between two parental species of tobacco, and six somatic hybrids produced as a result of fusion of vacuolated and evacuolated protoplasts, and subsequent culture. It was necessary to test a small number of commercially available ten- base synthetic oligonucleotide primers before arriving at a number which clearly distinguished the species and hybrids. The use of micro polyacrylamide gel electrophoresis coupled to silver staining for DNA provided a quick, reliable and very sensitive method of detecting polymorphisms. This procedures and protocols developed were applicable to parental species as well as to all hybrids tested, and using just four primers provided enough polymorphic bands to statistically distinguish all plants. The RAPD technology is a versatile, precise. Sensitive and cost effective method for genomic analysis of hybrids. Zhang et al (2005) found the polymorphism, similarities and relationships among Nicotiana tabacum L. cultivars were assessed with RAPD analyses. One hundred and

34 forty-nine bands were detected, of which 94 were polymorphic (63.1 %). A primer distinguishing all of the tested cultivars was found. High similarity between cultivars was revealed, and cultivar relationships were estimated through cluster analysis (UPGMA) based on RAPD data. Zhang et al., (2008). studied the genetic diversity among flue-cured tobacco cultivars. RAPD and AFLP analyses were used to assess the genetic similarity among selected accessions of flue-cured tobacco. Seventy eight RAPD and 154 AFLP polymorphic bands were obtained and used to assess the genetic diversity among 28 tobacco accessions. The cultivar relationships were estimated through the cluster analysis (UPGMA) based on RAPD data and AFLP data. The accessions were grouped into three major clusters and these shared common ancestry.

Tobacco (Nicotiana spp.) is one of the most important commercial crops in the world. During the last twodecades, molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The principles and characteristics of several molecular markers such as RFLP, RAPD, AFLP, microsatellites and minisatellites applied in tobacco genetics and breeding were reviewed. The application and development of molecular marker in tobacco genetic research was presented emphatically in the following areas: evolutionary genetics, population genetics and genotyping of cultivars, mapping and tagging of genes, and diversity analysis of germplasm (Liu and Zhang, 2008).

SivaRaju et al. (2009) demonstrated that the wild Nicotiana species are storehouses of genes for several diseases and pests, in addition to genes for several important phytochemicals and quality traits which are not present in cultivated varieties. Randomly amplified polymorphic DNA (RAPD) analysis was used to determine the degree of genetic variation in the genes Nicotiana and to develop species specific markers. 22 species and 2 interspecific hybrids were analyzed

35 by using 18 decamer primers. Greater amount of genetic polymorphism exists among the wild species of genus Nicotiana (99.5%) as evidenced by the high degree of polymorphism in RAPD profiles. The pairwise similarity measures in the species of subgenus Rustica was 0.0252 were as in the subgenus Tabacum it was 0.189 suggesting that there was significant diversity amongst the species of these subgenera. In the species of subgenus petunioides,the range of pairwise similarity measures was 0.128 to 0.941.The clustering pattern coincided with the traditional classification of Nicotiana species.All the primers generated specific bands in the various species.

36

I- Materials: This study was carried out in genetical lab.,Botany Dept., Fac. of Agric., AL- Azhar Univ., Cairo, and genetic engenering lab. and greenhouse , Dept. of Natural Products Res, National Center for Rad. Res. and Tec., Atomic Energy Authority, Naser City, Cairo.

A- Plant materials :- The seeds of Nicotiana alata was obtained from late Prof.Dr. A. H. Mohamed. Botany Dept., Fac. Of Agric., AL- Azhar Univ., Cairo.

B-Gamma irradiation source: 60Co was used a source of gamma rays with dose rate 1Krad/12min 15 sec. Nicotiana alata plantlets were exposed to gamma irradiation at National Center for Rad. Res. and Tec. Naser city, Cairo. C- Media: a- MS medium; Murashige and Skoog (1962) was used in this work and supplemented with defferent concentrations; Indol acetic acid (IAA) , Benzylaminopurine (BAP), Indole Butyric acid (IBA), Naphthalene acetic acid (NAA) or Kinetin (Kin mg/l) as follow for micro propagation: 1-MS+ 0.2 mg/l IAA + 0.2 mg/l KIN 2-MS+ 0.2 mg/l IAA + 0.5 mg/l KIN 3-MS+ 0.2 mg/l IAA + 1.0 mg/l KIN 4-MS+ 0.5 mg/l BAP + 0.1 mg/l IBA 5-MS+ 1.0 mg/l BAP + 0.2 mg/l IBA 6-MS+ 1.5 mg/l BAP + 0.3 mg/l IBA 7-MS+ 0.05 mg/l NAA + 0.2 mg/l IBA 8-MS+ 0.05 mg/l NAA + 0.5 mg/l IBA

37 9-MS+ 0.1 mg/l NAA + 0.1 mg/l IBA 10-MS+ 1.0 mg/l NAA+ 0.5 mg/l IBA 11- MS +2.0 mg/l BAP

b- MT medium; Murashige and Tuker (1969) medium was used in this work and supplemented with hormones, benzyl amino purine (BAP) and / or indol butric acid (IBA) as follow for micro propagation:

1- MT + 0.5 mg/l BAP + 0.1 mg/l IBA 2- MT + 1.0 mg/l BAP + 0.2 mg/l IBA 3- MT + 1.5 mg/l BAP + 0.3 mg/l IBA 4- 1/8 MT + 0.5 mg/l BAP + 0.1 mg/l IBA 5- 1/8 MT +1.0 mg/l BAP + 0.2 mg/l IBA 6- 1/8 MT +1.5 mg/l BAP + 0.3 mg/l IBA 7- 1/4 MT + 0.5 mg/l BAP + 0.1 mg/l IBA 8- 1/4 MT +1.0 mg/l BAP + 0.2 mg/l IBA 9-1/4 MT +1.5 mg/l BAP + 0.3 mg/l IBA 10-1/2 MT +0.5 mg/l BAP + 0.1 mg/l IBA 11-1/2 MT +1.0 mg/l BAP + 0.2 mg/l IBA 12-1/2MT+1.5 mg/l BAP+0.3I mg/l BA

c- N medium; Nitsch (1977) was supplemented with or without hormones, naphthalene acetic acid (NAA) and / or Kinetin (Kin) as follow for micro propagation.

1-N 2- N + 0.2 mg/l NAA + 0.5 mg/l KIN 3- N + 0.5 mg/l NAA + 0.5 mg/l KIN 4- N + 1.0 mg/l NAA + 0.5 mg/l KIN 5- N + 1.0 mg/l NAA + 1.0 mg/l KIN 6- N + 0.1 mg/l NAA

38 7- N + 1.0 mg/l KIN d-Callus Culture Media; a-Callus induction media MS medium supplemented with different hormones concentration / l as follows;

1- MS + 0.1 mg 2,4-D + 0.1 mg BAP 2- MS + 0.2 mg 2,4-D + 0.2 mg BAP 3- MS + 0. 3 mg 2,4-D + 0.5 mg BAP 4- MS + 0.5 mg 2,4-D + 1.0 mg BAP 5- MS + 0.1 mg NAA + 0.2 mg KIN 6- MS + 0.3 mg NAA + 0.4 mg KIN 7- MS + 0.4 mg NAA + 0.5 mg KIN 8- MS + 0.5 mg NAA + 0.5 mg KIN b-Regeneration media MS medium contaned BAP and NAA atdiffrerent concentration as;

1-MS + 0.5 mg BAP + 0.1 mg NAA /l 2- MS+1.0 mg BAP + 0.2 mg NAA /l 3-MS +1.0 mg BAP + 0.5 mg NAA /l 4- MS+1.5 mg BAP + 0.5 mg NAA /l 5- MS +1.5 mg BAP + 1.0 mg NAA /l 6- MS + 2.0 mg BAP /l

II- Methods: A- Callus subculturing and haploid induction; Medium size of flowering buds cutts were washed with tap water then soaked in clorox (10 % at 10 min ), and then washed three times in sterile bidistilled water to assure that any residues of clorox had been removed . The anthar were extracted from flowers before tetrad stage in laminar flow . The anthar were placed in jars contained MS

39 medium + 0.2 mg IAA /l +.05 mg Kin /l+ 30 gm sucrose / l + 8.0 g/l agar at pH 5.8.The cultured jars were carried out to growth chamber at 25 ± 2°C under photoperiod 16h of 3000 lux intensity.

C- Colchicine treatments: Dihaploid buds Nicotiana alata plantlets were cultured on MS media containing 0.2 IAA mg/l + 0.5 KIN mg/l and colchicine solution with different concentrations (0, 0.07, 0.09 and 0.1 % (w/v). The cultured jars were carried out to growth chamber at 25 ± 2°C under photoperiod 16h of 3000 lux intensity for 40 days before transfer to the pots. Diploid plants were obtained from the plants treated with the lowest concentration of colchicine. The plants which treated with colchicin were tested morphologically and cytological.

C-Gamma irradiation treatments: The flowers buds were irradiated with gamma radiation doses 0.0, 2.5, 5 , 7.5 , 10 , 15 , 20 and 25 Gy. The buds were strilized and anther were extracted and cultured in jars contained MS medium + 0.2 mg IAA +0.5 mg Kin + 30 gm sucrose + 8.0 g agar /l at pH 5.8. The cultured jars were carried out to growth chamber at 25± 2°C under photoperiod 16h of 3000 lux intensity.

D- Salinity treatments: Nicotiana alata explant (shoot) were cultured on MS media supplemented with 0.2 mg IAA + 0.5 mg /l Kin/l and containing of different NaCl concentrations (0. 0, 50, 100 ,150 and 200 mM). Cultured jars were incubated at the some condition subculturing were carried out after 4-5 weeks E- Combined effects between gamma radiation and salinity: Nicotiana alata explant (shoot) were exposed to combined effects between different gamma radiation doses 0, 10, 15, 20 and 25 Gy and NaCl concentrations 0.0, 50, 100 , 150 and 200

40 ml mol. Cultured jars were incubated at the previous conditions subculture were carried out after 4-5 weeks

F-Adaptation: The haploid plantlets explants were cultured on (MS + 0.2 mg IAA + 0.5 mg Kin/l). Cultured irradiated haploid plantlets with doses ( 0.0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy) on(MS + 0.2 mg IAA + 0.5 mg Kin/l). All this treatments were adapted to transfer to soil. Rooted shoots were washed from agar in running water. The plantlets at 5 cm high were transferred to jars containing sterilized clay. The plastic caps removed and replace with boring plastic sheets. After one week remove the plastic sheets and left to under the growth chamber or condition until to transferred to permanent pots. The permanent pots contain soil (peat moss to sand by ratio 1:1) as shown in Fig.1. The plantlets were transferred to greenhouse after two weeks. Data of shoot length and bud survival, were recorded after (40, 60 and 80 day).

Seeds cultured in green house to obtain on diploid plants directly. Then compared to distingwish between haploid irradiated haploid and diploid plants through size of flower, floral buds length, leaf aera,and polen grain.

1 2 3 4

Fig.(1) Adaptation stage; 1) 7 days, 2) 14 days, 3) 21 days and 4) 28 days.

41 G- Microspores examined:

The donor plants were collected and judged to be at the optimum stage. Healthy andvigorously growing flower buds before flowering when the microspores were estimated to be at the uninucleate stage. The exact stage of pollen grain development was also ascertained by a cytological test as Orhan and Garth (1998). Anthers were dissected from one flower bud of each cultivar, the anther length measured using a binocular microscope and then squashed in a drop of acetocarmine stain and the microspores examined under the microscope.

H-Chemical Analysis:

1- Estimation of proline content: Proline was determined using the method of Bates et al (1973). a) Extraction buffer 3% aqueous sulfosalicylic acid: b) Reagents: The mixture of acid ninhydrin (1.25g), glacial acetic acid (30ml), and Phosphoric acid (20ml) was warmed with agitation until dissolved then kept cool at 4ºC.

2-Estimation of Protein content: Bradford technique, (Bradford, 1976) for estimation of proteins makes use of the fact that the dye, Commassie Brilliant Blue G-250 (CI42655), undergoes a spectral shift in its of sorption maximum from 465 to 595 nm, when bound to the free amino groups of proteins .Its advantage lies in the fact that it is rapid, one step reaction with a fairly wide linear range as follows: 3- Molecular Markers

A- SDS- PAGE electrophoresis

42 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method described by (Laemmli, 1970) and modified by (Studier, 1973) as follow:

A) The monomer solution 29.2 g acrylamide and 0.8 g Bis-acrylamide were dissolved in 50 ml distilled water and completed to 100 ml, filtered and kept at 4ºC in a dark bottle to be used within three months.

B) Resolving gel buffer (1.5 M Tris -base, pH 8.8) 18.2 g Tris- base was dissolved in 50 ml distilled water and completed to 100 ml. The pH was adjusted at 8.8 with analar HCl.The solution was filtered and kept at 4ºC.

C) Stacking gel buffer (0.5 M Tris- base, pH 6.8): Six g Tris-base were dissolved in 50 ml distilled water and completed to 100 ml. The pH was adjusted at 6.8 with analar HC1. The solution was filtered and kept in a dark bottle at 4 °C.

D) Sodium Dodecyle Sulfate (SDS) 10%: Ten g SDS were dissolved in 50 ml distilled water and completed to 100 ml. This solution should be kept at room temperature to avoid precipitation.

E) Ammonium persulphate (10%): (initiator) 0.5 g ammonium persulphate was dissolved in 2 ml distilled water. This solution must be freshly prepared to avoid disactivation of the ammonium persulphate as a polymerization catalist.

F): N, N, N, N tetramethylenediamine (TEMED): TEMED is a strong polymerizing agent. The volume required to carry out polymerization depends on the quality of the used TEMED. In the present work, 50 µl and 30 µl of TEMED were added for stacking and resolving gels, respectively.

43

G) Overlay isopropyl: 50 ml of isopropyl alcohol and 5 ml distilled water.

H) Tank buffer: Six g Tris-base, 28.8 g glycine and 20 ml of solution (D) were dissolved in 500 ml distilled water and completed to 2 liters.

I) Staining-solution (1% COBB) Two g of coomassie brilliant blue (COBB, R-250) were dissolved in 200 ml distilled water, filtered and kept as a stock solution. The staining solution was prepared as follows: 62.5 ml of stain stock, 250 ml of methyl alcohol and 50 ml of glacial acetic acid were added, and then completed to 500 ml with distilled water.

J) Destaining solution: 45 ml methyl alcohol, 10 ml glacial acetic acid and 45 ml distilled water were added from the destaining solution.

K) Protein marker: A Sigma color marker consists of mixture of proteins. These proteins provide a visual monitor of protein migration during electrophoresis.

Extraction of Proteins:

Nicotiana alata was fine to powder using a pestle and a mortar in liquid nitrogen.Total soluble proteins were then extracted by mixing l g of this powder with 2 ml of samble buffer (1.21 gm Tris / HCl adjust to pH: 8.0 , 5gm sucrose or 5 ml glycerol, 1 ml SDS, 0.5 ml Betamercapto ethanol, then add bidistilled H2O to 50 ml) , vortexed and centrifuged at 10,000 rpm for 15 minutes. The clear supernatant was taken as the total protein extract.

44 Simple protein determination: Simple protein were analyzed by SDS- PAGE technique according to (Laemmli, 1970) currently the most commonly used electrophoretic technique in protein analysis due to the ability of the strong anionic detergent SDS, when used in the presence of disulfide bond cleaving reagents, to solubilize, denature and dissociate most proteins to produce single polypeptide chains. Here, the protein migration will be according to the molecular mass size only. Preparation of protein samples: Protein samples were prepared by mixing clear supernatant with the sample buffer (extract buffer) [Tris-HCI pH 6.8 , 2 % (w/v) SDS, 10% (w/v) sucrose, 0.1% (v/v) 2,β- mercaptoethanol, 0.5% (w/v) bromophenol blue] in the ratio of 1:1 and denatured by heating in a boiling water for 4 minutes, then loaded in equal amounts. For the native gel, protein samples were prepared by mixing clear supernatant with the treatment buffer, which in this case contains all components of the denatured gel treatment buffer except SDS (Sayed et al., 2004).

Gel preparation:

A- Resolving gel: Ten ml of monomer solution (soln. A), 7.5 ml of resolving gel buffer (soln. B), 0.3 ml of 10% SDS (soln. D), and 12 ml distilled water were mixed and shaked well. 300 µl of freshly prepared ammonium persulphate (soln.E3) were added and shacked well. Finally, 30 µl TEMED were added just before gel casting.

B- Stacking gel: 1.33 ml of monomer solution (soln. A), 2.5 ml of stacking gel buffer (soln. C), 0.1 ml of 10% SDS (soln D), 100 µl ammonium persulphate (soln. E) and 6.1 ml distilled water were added, respectively and shacked just before gel casting, 50 µl TEMED were added.

45

C- Gel casting: Biometra multigel-long is the device which was used in protein electrophoresis. It is a complete system with two sets of 12.5x 11 cm glass plates. One of the glass plates is a notched one with straight edge while, the other is with fixed 1.0 mm spacers. A silicon rubber seal, 1 mm was placed around the periphery of the fixed spacers, then, the other notched glass plate was carefully placed upon it. The two plates were added together by the mean of 6 clips. Two combined plates were prepared as above to carry out gel casting.

Resolving gel was poured in-between the two plates leaving 2 cm beneath the plates end. A layer of 90% isopropyl alcohol was added over resolving gel to prevent corrugation of the gel surface. Polymerization of the resolving gel took from half to one hour after gel solidification, which can be significant by the interphase line between the alcohols arid the solid gel, the alcohol was poured off.

Stacking gel mixture was added over the solid resolving gel till the top of the glass plates. A comb consisted of 12 wells and 1.0 mm derision was used. The comb was placed very gently into the liquid gel to avoid any air bubbles.

Stacking gel needed a pit longer time than resolving to be solidified. Once the solidification took place, the comb, clips and the silicon rubber were removed. The two plates Acre installed in the electrophoretic chamber. The tank buffer was added to immerse the wells completely. The protein samples were pipetted in wells by automatic variable micropipette (2-200 µl).

Gel electrophoresis:

The run was caried out at 30 V till to loaded samples passed the stacking gel and entered the resolving gel, then, the

46 volt was raised to 70 until the tracking dye (bromophenol blue) reached the gel bottom.

Staining and destaining of gels: The gels were stained for 24 hours in the prepared staining solution (1% COBB, R-250). To obtain a clear background, the gels were destained by the prepared destaining solution.

Photographing and gel scanning: Destained gels were photographed and the results were analyzed by the GDS (Gel docurnentahon system).

B-Randomly amplified polymorphic DNA (RAPD) a. DNA Extraction Young and fresh leaf samples were collected separately from 10 plants. All the selected leaves were normal and free from any pathogenic symptoms and all leave samples were saved in ice box and quickly transported to laboratory. Plant tissues were ground under liquid nitrogen to a fine powder, then bulked DNA extraction was performed using DNeasy plant Mini Kit (QIAGEN) (Tao et al. 1993). b. Polymerase Chain Reaction (PCR). PCR amplification was performed using ten random 10 mer arbitrary primers synthesized by (Operon biotechnologies, Inc. Germany) table (1) with the following sequences: Table (1 ): List of the primer names and their nucleotide sequences used

No Name Sequence 1 OP-C13 5` AAGCCTCGTC 3` 2 OP-D07 5` TTGGCACGGG 3` 3 OP-L15 5` AAGAGAGGGG 3 4 OP-L12 5` GGG CGG TAC T 3` 5 OP-M20 5` AGGTCTTGGG 3`

47 Amplification was conducted in 25 µl reaction volume containing the following reagents: 2.5 µl of dNTPs (2.5 mM), 2.5 µl Mgcl2 (2.5 mM), and 2.5 µl of 10 x buffer, 3.0 µl of primer (10 pmol), 3.0 µl of template DNA (25 ng / µl), 1 µl of Taq polymerase (1U/ µl) and 10.5 µl of sterile dd H2O. The DNA amplifications were performed in an automated thermal cycle (model Techno 512) programmed for one cycle at 94º C for 4 min followed by 45 cycles of 1 min at 94º C, 1 min at 36º C, and 2 min at 72º C. the reaction was finally stored at 72º C for 10 min. Amplified products were size-fractioned using ladder marker (100 bp +1.5 bp) by electrophoresis in 1.5 % agarose gels in TBE buffer at 120 V for 1 h. the bands were visualized by ethidium bromide under UV florescence and photographed.

48 I- Induction of haploid anther callus; Induction of haploid callus from anthers were studied on some media; Ms, MT& N; supplemented with different hormones concentrations; BAP, KIN, IAA, IBA and / or NAA; Results obtained were presented in tables; (1), (2) and (3). A-On MS medium; Cuts of anthers (25pices) were cultured on MS medium supplemented with different hormones concentrations, plantlets were tested for haploidy, Table (1) presented results obtained. Results in this table showed clearly that the best medium which contained 0.2 mg IAA + 0.5 mg KIN / l (20 plantlets) fallowed MS + 0.1 mg NAA + 0.1 mg IBA / l (16 plantlets), the haploid plantlets were ranged between 13 to 17 plantlets in all the rest treatments. MS medium supplemented with; 0.2 mg IAA + 0.5 mg KIN / l, 1.5 mg IBA+ 0.3 IBA / l or 1.0 mg BAP + 0.2 mg IBA /l were failed in rooting formation. On the other hard, MS medium supplemented with; 0.2 mg IAA + 0.2 mg KIN, 0.5 mg BAP + 0.1 mg IBA or 0.05 mg NAA + (0.2 or 0.5 mg ) IBA / l were gave; 16 , 16 , 17 or 14 plantlets; respectively; without callus formation. Callus morphology was only friable when 0.2 mg IAA + 0.5 mg KIN / l were added to MS medium. B- On MT media; Results obtained from anther culturing on MT media were presented in table (2), these results showed that complete, ½, ¼ or 1/8 MT medium were used in culturing haploid plantlets by supplementation with; (0.5, 1.0 or 1.5 mg) BAP+ (0.1, 0.2, or 0.3 mg) / l. Root formation were resulted by culturing on the MT 1 mg BAP + 0.2 mg IBA or 1/8MT + 1.0 mg BAP + 0.2 mg IBA / l, ¼ or ½ MT + 0.5 mg BAP + 0.1 mg IBA / l or ½ MT + 1.5 mg BAP + 0.3 mg IBA / l. The ¼ MT + 1.0 mg BAP + 0.2 mg IBA /l was failed to from roots or callus. On the other side, all the rest treatments were gave massive greenish callus.

49

Table (1): The effect of some hormones combination with MS medium on 25 anthers to produce haploid plantlets in Nicotiana alata;

NO. Plant growth haploid Root Callus Callus regulators Plantlets formation formation morphology ( PGRs ) / mg /l No 1 MS +2 IAA+0.02 16 - - - KIN 2 MS +0.2 IAA+0.2 16 16 - - KIN 3 MS +0.2 IAA+0.5 20 20 20 Friable KIN 4 MS+ 0.2 IAA+1.0 13 - 13 Massive KIN greenish 5 MS + 0.5 BAP + 0.1 16 16 - - IBA 6 MS + 1.0 BAP +0.2 14 - 14 Massive IBA greenish 7 MS +1.5 BAP + 15 - 15 Massive 0.3IBA greenish 8 MS +0.05NAA+ 0.2 17 17 - - IBA 9 MS +0.05NAA+ 0.5 14 14 - - IBA 10 MS +0.1NAA+ 0.1 16 16 16 Massive IBA greenish 11 MS +1.0 NAA+ 0.5 15 15 15 Massive IBA greenish

50

Table (2): The effect of some hormones combination with MT medium on 25 anthers to produce haploid plantlets in Nicotiana alata .

haploid Root Callus No Plant growth regulators Plantlet formation Callus morphology . (PGRs ) / mg /l formatio formation

n MT + 0.5 BAP + 0.1 IBA 9 - Massive 1 9 greenish MT + 1.0 BAP + 0.2 IBA 12 12 2 - -

MT + 1.5 BAP + 0.3 IBA 11 - Massive 3 11 greenish 1/8 MT + 0.5 BAP + 0.1 IBA 13 - Massive 4 13 greenish 1/8 MT +1.0 BAP + 0.2 IBA 10 10 5 - -

6 1/8 MT +1.5 BAP + 0.3 IBA 12 Massive 12 - greenish 1/4 MT + 0.5 BAP + 0.1 IBA 11 7 - - 11 1/4 MT +1.0 BAP + 0.2 IBA 11 - 8 - -

1/4 MT +1.5 BAP + 0.3 IBA 8 - Massive 9 8 greenish 1/2 MT +0.5 BAP + 0.1 IBA 10 10 10 - -

1/2 MT +1.0 BAP + 0.2 IBA 9 - Massive 11 9 greenish 12 1/2 MT +1.5 BAP + 0.3 IBA 9 9 - -

51 C- On N medium; Results obtained from Table (3) showed that the anther cultures on N medium containing 0.2 mg NAA + 0.5 mg KIN / l were 11 haploid plantlets, also N medium + 1.0 mg KIN / l was gave 11 haploid plantlets with rooting formation only. All N medium supplemented with; 0.1 mg IAA / l , 0.5 mg from each NAA and KIN / l , 1.0 mg NAA + 0.5 or 1.0 mg KIN / l or 1.0 mg from each NAA and KIN were gave plantlets, Root and callus. Only N medium + 0.5 mg from each NAA and KIN / l was friable. Finally, the best medium from all tested to give plantlets; root formation and friable callus formation was MS supplemented with 0.2 mg IAA + 0.5 mg KIN / l, the anther culture plantlets were tested for the ploidy level , results from chromosome number in root tips revealed that they are haploid (9 chromosomes).

Table (3): The effect of some hormones combination with N medium on 25 anthers to produce haploid plantlets in Nicotiana alata .

Plant growth haploid Callus Callus No. regulators Plantlet Root formation morphology (PGRs ) / mg /l formation formation N + 0.2 NAA + 0.5 KIN 11 - 1 - -

N + 0.5 NAA + 0.5 KIN 9 9 2 9 Friable

N + 1.0 NAA + 0.5 KIN 7 7 Massive 3 7 greenish N + 1.0 NAA + 1.0 KIN 11 11 Massive 4 11 greenish N + 1.0 KIN 11 11 5 - -

6 N + 0.1 IAA 9 9 Massive 9 greenish

52 The production of haploids has been tried in quite a few species since the late 1920,s by pollinating the target plant with the pollen of distantly related species or inactivated pollen (Kostoff, 1929 and Katayama, 1934). However, these efforts yielded no efficient and reliable method usable in haploid breeding until Guha and Maheshwari (1964) succeeded with an anther culture of Datura. Since then, haploids have been obtained efficiently in many species by anther culture. Nitsch (1969) reported the first production of haploid plants through anther culture of Nicotiana alata. The majority of pollen plants in tobacco are haploid (Bourgin et al., 1979). Meanwhile Nitsch (1969) observed that androgenesis in most Nicotiana sp. follows the pathway of embryo formation. The shift of development from embryo to callus in anther culture of Nicotiana sp. May be attributed to presence of 0.1 mg/l NAA in the culture medium but a weaker auxin (0.1 mg/l IAA). Chu (1978) cited that the N6 is a basal medium for callus induction to anther culture of cereal crops and Murashige and Skoog (MS) as a basal medium to regenerate plants. Also Chaturvedi and Sharma (1985) indicated that culturing of anthers with uninucleate pollen grains at the tetrad stage on modified Murashige and Skoog medium supplemented with 0.5 mg /l BA and 1 mg / l NAA for 20-30 days differentiated into embryoids. Ling et al., (1990) found that the embryogenic callus was successfully maintained through sub culturing on solidified MT medium supplemented with 185 µM adenine sulfate and 2.8 µM gibberellic acid (GA3). Oh et al., (1991) clarified that 0.5 mg/l IBA either with or without 0.1 or 0.5 mg/l BAP was the best concentration for rooting of Citrus junos. Yang and ludders (1994) reported that MS medium supplemented with 0.5 mg/l BAP and 0.1 mg/l IBA resulted in significant better shoot formation of pistachio Rahaman et al., (1996) declared that ovules of 5 citrus species cultured on MS medium supplemented with either furfuryl aminopurine, Kinetin or BAP (0.0, 0.25, 0.5, 0.75 1.0 and 1.25 mg/l) developed into cotyledonary embryiods. Eugenio-Perez (1997) stated that Murashige and Skoog medium supplemented with B5, vitamins, 5% sucrose, 33.3 µM

53 BAP and 5.4 µM NAA is the optimal culture medium for both species of Mexican lime and mandarin. Singh et al., (1994) found that the best multiplication of citrus was obtained when MS medium supplemented with 1.0 mg/l BAP, 0.5 kinetin and 0.5 mg/l NAA. Maggon and Singh (1995) clarified that adding 2 mg/l BA to MS medium had a primitive effect on the number of sweet orange shoots produced. Koc et al., (1992) found that Murashige and Tucker (MT) medium was the most suitable for culturing of Shamouti orange ovules. The obtained callus developed into the greatest number of embryoids when subcultured on White medium contained 1.0 mg /l BA and 0.5 mg /l NAA.

54 II-Haploid anther callus Induction; Anther cultures were used as a source of haploid plants, anthers were cutted through mitotic deviations and dehaploid to pollen greains, piceas were transplanted on MS medium with different hormones concentrations; (2,4-D, KIN, NAA and / or BAP). Callus were examined cytologically for haploidy after 40 days, callus formation percentages, sizes and morphology were presented in table (4). Data in this table showed that MS medium with 0.4 mg NAA+ 0.5mg KIN/l was the highest formation percentage (58%) of haploidy callus and sizes (Fig.2B), while 0.2 mg 2,4-D+.02 mg BAP were the lowest (20%) for haploidy callus formation and sizes(Fig. 2A).

III-Regeneration of haploid plants;

Haploid calluses were subcultured on MS+ 0.4 mg NAA + 0.5 mg KIN/l, then transfered to a new Jars contained MS medium supplemented with different ratios from BAP: NAA for regeneration,obtained results after 40 days were presented in table(5) and Fig. (3). Results in this table showed clearly that the addition of; 0.5 mg BAP + 1.0 mg NAA,1.0 mg BAP + 0.2 or 0.5 mg NAA or 1.5 mg BAP + 0.5 mg NAA were succeeded in regeneration. The MS + 1.0 mg BAP + 0.5 mg NAA was more effective to have plantlets (33%). On the other hand MS with; 1.5 mg BAP + 1.0 mg NAA or 2 mg BAP were failed to regenerated and plantlets formation.

55 Table (4);Percentages of haploidy formation, size and morphology for anther calluses on MS medium supplemented with different hormone concentrations after 40 days on 25 °C.

No. Hormones(mg/l) Callus Callus Callus formation% size morphology

1 MS + 0.1 2,4-D + 0.1 BAP 31 small Massive greenish

2 MS + 0.2 2,4-D + 0.2 BAP 26 small Massive greenish

3 MS + 0.3 2,4-D + 0.5 BAP 51 medium Friable

4 MS + 0.5 2,4-D + 1.0 BAP 44 small Massive greenish

5 MS + 0.1 NAA + 0.2 KIN 39 small Massive greenish

6 MS + 0.3 NAA + 0.4 KIN 36 small Massive greenish

7 MS + 0.4 NAA + 0.5 KIN 58 large Friable

8 MS + 0.5 NAA + 0.5KIN 49 medium Friable

56

A B

Fig. (2): haploid Nicotiana alata anther callus on MS medium supplemented with; (A) 0.2 mg 2, 4-D + 0.2 mg BAP/l and (B) 0.4 mg NAA + 0.5 mg KIN/l after 40 days.

57 Table (5): The combined effect of BAP+NAA hormone concentrations on plantlets regeneration in haploid calli for Nicotiana alata after 40 days on 25 °C. No. Media + hormone Regeneration (mg/l) Plants No. 1 MS + 0.5 BAP + 0.1 NAA 10

2 MS + 1.0 BAP + 0.2 NAA 16

3 MS + 1.0 BAP + 0.5 NAA 33

4 MS + 1.5 BAP + 0.5 NAA 12

5 MS + 1.5 BAP + 1.0 NAA -

6 MS + 2.0 BAP -

A B C D

Fig. (3): Plant regeneration N. alata. on MS medium supplemented with different BAP&NAA concentrations after 40 days;

A) MS + 0.5 BAP + 0.1 NAA B) MS + 1.0 BAP + 0.2 NAA C) MS + 1.0 BAP + 0.5 NAA D) MS + 1.5 BAP + 0.5 NAA

58 Many workers were studied the anther calluses and plant regeneration in solanaceae family, the genetic factors were involved to reveal the haploid calluses induction and plant regeneration in this family. Genetic factors are involved in determining the response to anther culture as frequency of monohaploids could be promoted by selection in potato crosses between responding and also non-responding parents (Jacobsen and Ramanna, 1994). F1 hybrids between poor and fairly responsive pepper parents showed fair responses (Mityko et al., 1995). Tomato cultivars containing the male sterility gene ms 1035 showed strong response to anther culture for both callus induction and regeneration of plants (Zagorska et al., 1998). More studies are needed to reveal the genetic mechanisms for haploid induction by androgenesis. In vitro anther cultures technique in all important crops were investigated through the last 30 years, Nichterlein et al. (1989 and 1991 ) in Linum usitatissimum anthers were improved the technique for obtaining a larg number of haploid plants to using in plant breeding and selections, they also studied the genotype of pretreatment on callus formation and differentiation.Objectives of the present studies were to assess the possibility of anther cullure of linseed as a method of haploidy production, establish the effect of genotype and pretreatment on anther cultre induction and plant regeneration on solid or liquid medium. Zhang (1996) reported that in anther culture, the different rice genotypes had different callus induction and callus differentiation into plants. The efficiency of anther culture was based on number of green plants and double haploid plants produced either spontantly or induced. The production of green plants through anther culture was dependent on the response of the anthers under in vitro culture as well as success in the callus induction and callus regeneration into the green plants. Abilities to induce callus or embryoid growth from microspore or pollen are therefore the main factors in influencing the efficiency of anther culture (Zhou, 1996). The first callus initiated was generally very easily differentiated into green plants (Sasmita et al., 2001 and Dewi et al., 2004). Orhan and Gareth (1998) cultured anthers are expected to by pass their normal pathway of development with the result that

59 individual pollen grains divide repeatedly to form a callus. After five weeks of implantation, the initiation of growth varied from anther to anther. With time, the growth increased forming a distinct callus tissue of irregular shape and size. This phenomenon was observed until the ninth week of culture in some of the anthers. The color of the callus varied from creamy white, creamy to light yellow. The texture was smooth or rough and loose or compact. Afterwards some of the calli became brown and then black and finally died.Results obtained showed that the untreated callus (control) can be differentiated to shoot and root. These results agree with many investigators; Lam (1975) reported the induction of embryoid bodies and shoots from potato tuber callus grown on a medium containing MS salts,. The addition of BA to MS medium was essential for shoot induction from somatic embryos. Roest and Bokelmann (1980) regenerated shoots from callus formed from the cut rachises of compound leaves cultured on a medium MS medium containing BA, IAA and GA as the growth regulators. The addition of BA and GA to the NAA- containing medium resulted in dramatic increase in the number of shoots produced per shoot-tip callus. Plants have also been regenerated from leaf calli of dihaploid potato clones (Jacobsen, 1977).Utilizing a relatively short culture period, Roca et al., (1978) regenerated multiple shoots from merstim tip calli that did not vary leaf, flower, or tuber morphology, or in chromosome number.Organogenesis has also been induced in sweet potato through somatic embryogenesis using shoot tips and other explants, while most of these studies were aimed at plant regeneration in sweet potato, the frequencies of regeneration reported are not high. (less than 1% and required about 5 months to develop shoots). Our studies were encouraged by the initial results obtained with the two step protocol for sweet potato regeneration using leaf explants described by Medina (1991) which was adapted to develop an improved system for regeneration of adventitious plants in sweet potato.

60 IV-The haploid confirmation; Plantlets obtained from farther studies were grown to be flowering plants, then they examined morphologically and cytologecaly compaired with their diploid parental plants, haploid plants were found to be less size for leaf,stem diameter and growth vigour. While, plant length and flower size were differ into less and equal, alsoflower color was the same (Fig.4).Haploid plants were flowered lately, these flowers were less in number / plant and anther number /flower, the haploid anthers were almost empty from the third layer, the standing squshos of root tips by acytocarmine staine gave the chromosome number in each haploid and diploid plants(Fig.5a & b), while the staining of pollen grain in haploid plants showes the empty of anthers except from some dipress comparing with diploid pollen grains (Fig.6a & b)

D H

H D D H (a) (b)

D H D H D H

(c) (c1) (c2)

Fig. (4): Morphological differences between Diploid parent and haploid plants; (a) in plant size, (b) Leaf size (c) Flower size (c1) and anthers(c2)

61

(b)

(a) Fig. (5); chromosome number in; (a) diploid plant root tip (18 chromosomes) in metaphase of mitosis.(b) haploid plant root tip (9 chromosomes) in metaphase of mitosis.

nd

Fig. (6): Stained pollen grain in ;( a) diploid plants (b) haploid plants

62 V-Diplodization of haploid lants.

Cuttings of haploid plants were culture on MS medium supplemented with 0.2 mg IAA + 0.5 mg KIN /l and containing different colchicin concentration (0.07, 0.09 and 0.1%). Growing plants were examined cytologicaly for haploidy, and results obtained showed the failing of each of; 0.09% or 0.1% colchicin dose to diploidizatation of haploid plants, while 0.7% colchicin was succeded to be a diploid plants and gave some flowers (Fig.7), which gave some seeds when pollinated with parental diploid plants. Fig. (7) also presented the diploidizatation pollen grains in colchidiploid plants. Androgenesis is the process whereby an embryo is developed from a microspore. Microspores are haploid cells formed by meiosis. In the normal process, microspores undergo an unequal mitotic division leading to the formation of a generative and a vegetative nucleus. The generative one divides one more time to form two sperm nuclei, either before or after pollen grain maturation. Androgenesis as induced by anther culture alters the development process of microspores such that the first mitosis is often symmetrical in mode. This leads to the formation of two identical nuclei in the cells that develop into embryos or callus. Plantlets are then generated either directly from the embryos or indirectly by inducing roots and shoots from the callus. The various techniques for anther culture and induction of androgenesis are summarized by Chlyah et al., (1990) and Summers (1997) for tomato and Vagera (1990) for pepper. A number of factors affect the success of anther culture. Attempts on dipldization through leaf culture of haploid plants by Mercado et al., (1985) and Beltran et al., (1986) gave only 1.3% and 2.0% doblling, respectively. Reserarch efforts abrots broad towards restoring diploidy of N. tabaccum haploids were more successful, that is, 33 to 67 % doubling was observed (Kasperbauuer and Collins, 1972 and Collins et al., 1974) Have also explored the application of colchicine to induce chromosome doubling in haploid plants. The low survival rate may be due to the inadequacy of the growing conditions for the

63

(60×) (a) Diploid

(120×)

(b) Dihaploid

Fig. (7): flowers and pollen grains in;(a) diploid and (b) dihaploid plants

64 treated materials, that will help prime the meristematic cells for doupling and overcome the growth retardation that immediately follows colchicine treatment. The induction of tetraploid plants in Nicotiana alata indicated that colchicine is an anti-mitotic agent which inhibit the formation of spindle fibers and effectively arrest mitosis of at the metaphase stage leading to polyploidy (Hancock, 1997) by binding to the tubulin dimmers, preventing the formation of microtubules and consequently, spindle fibers during cell division (Petersen, et al., 2003). So, the chromosome have been where they duplicated and obtained tetraploid plants of Nicotiana alata. Similar results indicated various responses to Joshi and Rakesh, (2004) colchicine in different species or varieties. Morphological comparison between haploid and diploid plants showed some differences in both vegetative and flower characteristics. The morphological changes of tetraploid plants were highly significant than the diploid plants in stem length, number of leaves on plant at flower initiation, leaf breadth, leaf length, leaf internodes number and leaf internodes length. The tobacco species N. tabacum L. is of amphidiploid origin (2n = 48) (Smith, 1979). and meiotic behaviour in its androgenic haploids was shown to be similar to monoploids (Emiro, 1980).Thus, haploids are sterile and in order to get seed their chromosomes have to be doubled. The successful application of haploid technology to plant breeding depends on both reliable methods for production of haploids in large number and achieving a high frequency of chromosome doubling. For dihaploid production, effective colchicine treatments have been reported for several crop plants like rapeseed (Weber et al., 2005) and Indian mustard (Prem et al., 2004). In some projects on Turkish tobaccos, colchicine has been applied to double the chromosome number. Emiro (1978) used this chemical to overcome interspecific hybrid sterility, Emiro et al. (1987)reported that besides acenaphthene, also used colchicine in a breeding project based on the haploid technique. In the former study Emiro (1978), treatment of seeds in an aqueous solution of colchicine has been reported to be more effective compared to germinated seed and axillary bud treatments. In the latter Emiro et al. (1987), colchicine was applied to growing points of haploid plantlets in pots and resulted in low chromosome doubling (11.97%).

65 VI- Radiation effect on haploid anther callus induction and regeneration; Small pices of haploid callus were irradiated with gamma rays and grawn on MS medium + 0.4 mg NAA + 0.5 mg Kin / l. Gamma rays doses were; 0, 5, 10,15and 20Gy, data obtained were taken after 40 days and presented in table (6), results in this table shows clearly that all doses were affected in callus sizes, while callus morphology were the same effect after 5 Gy (Massive greenish). On the other hand, the size of callus was gradually decreased by increasing the irradiation doses, also the regeneration was stopped by gamma rays.

Table (6): Effect of gamma radiation on haploid callus size, morphology and regeneration in Nicotiana alata after 40 days.

Gamma Mean of callus Callus Plant radiation size /Cm3 morphology regeneration doses /Gy 0 0.92 Friable + 5 0.79 Friable - 10 0.67 Massive greenish - 15 0.50 Massive greenish - 20 0.41 Massive greenish -

The principle site of the damage caused by ionizing radiations to plants in the cell nucleus. The effect on the callus growth, in terms of size or weight, which in used as the criterion of sensitivity to gamma radiation, shows variations between different species of higher plants as large as 500 fold (Sparrow and Woodell, 1962). These variations are correlated with chromosome numbers and nuclei with large volumes are highly sensitive. The rate of nuclear division also important. Sparrow et al. (1961) observed that thier was a lose correlation between the nuclear volume or DNA content and the radiosensitivity of plant meristem, that is the larger nuclear volume of DNA

66 content, the more sensitive the increase of callus growth is related to the increase in the mitotic rate. According it was considered that the species with activity growing calli were generally highly sensitive to radiation. Our results are shown that the increase in radiation doses (0, 5, 10, 15 and 20 Gy) induced decrease in anther callus size. This was in agreement with Mkuya et al. (2005) who indicated that, exposed rice anthers to radiation doses (0, 5, 10, 15 and 20 Gy) induced decrease in callus formation with the irradiation dose increased.Also, this is in line with Nakamura and Hattori (1997) who investigated the effect of 60Co gamma-ray irradiation at different culture stages on rice anther culture. Callus formation rates, plant regeneration and organogenesis decreased with increasing doses of radiation in rice anther culture. Zhao et al. (1983) irradiated anther-derived callus with gamma rays and compared the effect of irradiation dose on regeneration, they found that when the dose was above 0.6 Gy the regeneration frequency would decrease sharply. Chen et al. (2001) demonstrated that the dose of 20Gy gamma rays had significant stimulation effect on regeneration of green plants from rice anther culture. Kinoshita et al. (1989) irradiated anthers just after inoculation on the callus-forming medium or anther-derived calli with gamma rays at acute or chronic irradiation rates. They found that the rates of green-plant regeneration were reduced under different irradiation treatments, except that from irradiated calli at the 500 R/min. Based on these results, in vitro mutagenesis of rice were used for selecting mutants with drought tolerance Khan (2001), salt tolerance Lee et al. (2003), herbicide resistance Bae et al. (2002), increasing contents of specific free amino acids Kim et al. (2004) .This method was also applied for anther culture of wheat, maize, oilseed, tomato, tobacco, petunia and apple etc.

67 VII- Radiation effect on haploid plant induction; Sterilized anther or buds were irradiated by; 0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy gamma rays, anthers or buds were extracted and planted in gars (5 anther or buds / gar) contained MS medium supplemented with 0.2 mg IAA , 0.5 mg KIN, 30.0 g sucrose and 8.0 g agar. Sample of 100 growing plants were tested for haploidy, the haploid plants number, shoot length and root formation were noticed and presented in tables (7) and (8). Table (7) showed cearly that haploid production percents were decreased by increasing gamma radiation dose, the same order were found in shoot lengths which decreased gradually by increasing radiation dose. This table determined also that the root formation was stopped after 2.5 Gy gamma rays. Table (8) presented gamma rays effects on haploid plants obtained from buds, the same traned were noticed as anther haploid plant survivals and shoot lengths, and also the root formation for bud haploid plants were gave the same trands. Fig (8) was presented data in table (8), it was showed the plants after 40 days from radiation treatments, we can noticed from this figure that the survival percentages were gradually decreased, (the lowest at 25 Gy and the highest at 2.5 Gy). These data were significant at L.S.D (P ≥ 0.42). Gamma rays are the most energetic form of such electromagnetic radiation as X- rays, visible light and UV, having the energy level from around 10 kiloelectron volts to several hundred kiloelectron volts. Gamma rays more penetrating than other radiation as alpha and beta rays (Kovács and Keresztes, 2002). Ertan Sait Kurtar et al., (2009) reported that Haploidization is the process of creating a haploid (n) cell, usually from a diploid (2n) cell. Haploid plants can be obtain spontaneously (androgenesis, gynogenesis or parthenogenesis, semigamy and polyembryo) or using with some haploidization techniques (in vitro androgenesis, in vitro gynogenesis and in situ parthenogenesis). These techniques facilitate the rapid production 68

Table (7): Effect of gamma radiation doses on 100 haploid plants preduced from Nicotiana alata anthers.

Mean of Mean plant number of; Root Radiation shoot formation dose/ Gy. Growin Haploid length

g /cm No. % No. %

+ 0 100 100 62 100 6.0 + 2.5 90 90 57 91.9 5.1 - 5 81 81 51 82.3 4.5 - 7.5 70 70 46 74.2 3.8 - 10 62 62 37 59.7 3.2 - 15 48 48 30 48.4 2.7 - 20 36 36 21 33.9 2.1 - 25 22 22 9 14.5 1.0

LSD. (5%) 0.57

69

Table (8): Effect of gamma radiation doses on 100 buds survival in Nicotiana alata.

Mean plant number of; Mean of Root Radiation shoot formation dose/ Gy. Growing Haploid length /cm No. % No. % 100 + 0 100 96 100 7.2 95 + 2.5 95 87 90 6.5 83 - 5 83 81 84 5.5 76 - 7.5 76 74 77 5.0 68 - 10 68 68 70 4.6 - 51 15 51 49 51 4.1 - 45 20 45 37 38 3.6 - 36 25 36 29 30 3.0

LSD. (5%) 0.42

70

Control 2.5Gy 5Gy 7. 5Gy

10Gy 15Gy 20Gy 25Gy

Fig. (8): Effect of gamma radiation doses on haploid plant survival in Nicotiana alata obtained from buds.

71

of pure lines from heterozygous plants in a single generation and represent advantages significant for breeders and geneticists. At the pollen irradiation (UV, gamma rays, X-rays) is the most widely used technique to induce in situ haploid plants. Gamma rays recommonly used in haploidy programmes because of their simple application, good penetration, reproducibility high mutation frequency and less disposal problems (Chahal and Gosal, 2002). This technique consists of the induction of in situ gynogenic embryos with gamma rays irradiated pollen and then followed by rescue of haploid embryos by in vitro culture. The results for radiation sensitivity test based on survival percentage of irradiated and non-irradiated plantlets demonstrated that significant reduction in survival percentage was observed with increasing gamma dosage. For the survival percentage of irradiated plantlets to reach 50%, the gamma dosage administered was 72.5Gy. These results were in accordance with the radiation sensitivity test done by Norfadzrin et al. (2007). Also, Sax, (1963) have shown that survival of plants to maturity depend on the nature and extent of chromosomal damage. Increasing frequency of chromosomal damage with increasing dosage may be responsible for less germ inability and reduction in plant survival and plant growth. The chemical and physical changes occurring in the biological applications under the influence of radiation must be thoroughly investigated to understand what happened to the physiological activity leading to generate genetic variation in the biological applications (Beloshapkina, 1998). Gamma rays belong to ionizing radiation and interact with atoms or molecules to produce free radicals in cells. These radicals can damage or modify important components of plant cells and affect differentially the morphology, anatomy, biochemistry, and physiology of plants depending on the irradiation level. These effects include changes in the plant cellular structure and metabolism, e.g., dilation of thylakoid membranes, alteration in photosynthesis and modulation of the antioxidative system (Kovács and Keresztes, 2002 and Kim et al., 2004).

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Norfadzrin et al. (2007) found that the increasing in gamma dosage decrease the germination percentage and survival percentage of tomato and okra. All plantlets whether subjected to various dosages of irradiation or non-irradiated, demonstrated 100% survival at the first week of culture. The survival percentage of irradiated plantlets dropped remarkably with increased dosage during the second and third week of culture. However, plantlets irradiated at low dosage 10Gy, were able to withstand the mutagenic effect of gamma ray and exhibited 100% survival for 3 weeks. The survival percentage of plantlets irradiated at the highest dosage, 70Gy, fell considerably from 100% to 55.5% at the second week of culture to 44.4% at the third week of culture resulting in a total decrement of 55.6%. Similar observations were made for plantlets irradiated at 20 to 60Gy whereby all of them displayed a gradual reduction in survival percentage corresponding to the increased in gamma dosage. Hiroshi et al., (1995) studied that the tobacco (Nicotiana tabacum ) anthers which had been cultured for (0, 10 and 20 days) were irradiated with gamma radiation doses (0, 2.5, 5, 10, 20, 40, 80 and 160 Gy). Anthers cultured for 10 days before irradiation, which were correspondence to a plantlet initiation stage of microspore, were most radiosensitive and RD50 for relative anthe cultue (RSCR) (defined as dose resulting in 50% reduction in relative anther culture response) was 8 Gy. On the other hand, RD50s for relative anther cultue (RACR) in the anthers precultured for 0 and 20 days were 50 Gy. The highest frequency of morphological variants was also obtained from the plants derived from irradiated anthers precultured for 10 days. Gamma irradiation on the anthers precultured for 10 days is most effective in inducing mutation in tobacco. Behnam et al. (2008) studied the effect of gamma irradiation in wheat anther culture contain auxins. Auxins were essential for the induction of callus, and the type of carbon source also affected the induction. Such a phenomenon is considered as a sort of recovery from irradiation damage but it can also be speculated that pre-irradiation of seeds at low doses would determine some morpho-physiological hanges that prime germination and growth. While negative

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effects on germination can be ascribed to abnormalities in mitotic cycles and in metabolic pathways of the cells, the increase in germination rate can be attributed to structural reasons. More specifically, Hammond et al. (1996) reported that long-term exposure to cosmic rays increases the porosity of the nutritional layers and teguments of tomato seeds, thus allowing the seeds to absorb water and nutrients more readily. As a consequence, seeds germinate easier and seedlings grow faster. In general, radiation exposure can induce both negative and positive effects on plants, and the mutations at the base of these effects can also be transmitted to the progeny However, the changes in agronomic characters, as well as the transmission of these and other morphological traits to the next generations, are dependent on the species and variety as demonstrated not only with experiments in laboratory but also with irradiation during spaceflight (Wu and Yu, 2001 and Yu et al. 2007). For example, after irradiation during a 10-day Spacelab mission, dry seeds of A. thaliana developed plants whose radicle was increased or decreased in length depending on the mutant (Kranz and Bork, 1984). The shortening of plant height has been measured only in some cultivars of maize and rice grown in space (Bayonove et al. 1984 and Yu et al. 2007) exposed 11 pure rice varieties to space radiations and compared the plants obtained sowing those seeds with the control plants, showing that radiation exposure induced the formation of taller plants with longer spikes and higher number of heavier seeds. However, sterility has been demonstrated to be reliant on LET and to be dose dependent (direct proportionality) in various species such as Arabidopsis, rice and maize (Shikazono et al. 2002). Apart from alteration in the meiotic cycle, sterility can be due to the development of plants unable to produce fertile floral elements such as anthers (Wu and Yu, 2001). In Spinacia oleracea, irradiation of seeds with high doses of carbon ions constrained germination and growth up to the development of aberrant flower stalks, while no significant effects were found after irradiation with helium ions (Komai et al. 2003). Moreover, in this species, the same authors demonstrated the occurrence of a sexual modification versus the

74

expression of andromonoecy induced by the exposure to ion irradiation. The presence of abnormalities in fruits and the formation of defective ovules are considered a very sensitive parameter to evaluate the effect of ionizing radiations as reported in A. thaliana (Zimmermann et al. 1996). The exposure of plants to ionizing radiations determines changes of the genetic material that are responsible for the alteration in mitotic and meiotic capacity of cells, thus resulting in modification of growth and reproductive processes. In plants, also totipotency, hence morphological differentiation, is affected by radiation exposure. On the other hand, radiations have an important role in the induction of mutations that are revealed through various changes in the phenotype. Many studies have been carried out to evidence the effects of high radiation levels on the mitotic/meiotic ability, totipotency and differentiation of plant cells, through the analysis of various parameters such as germination and survival rate, lethality, organ differentiation, tumourization, occurrence of morphological aberrations, flowering rate and sterility (Hirono et al. 1970; Tanaka et al. 1997; Hase et al. 1999; Wu and Yu 2001; Durante and Kronenberg, 2005 and Zhou et al. 2006). As for mutational analysis, available results are not always easily comparable mainly because of the utilization of different methodologies during irradiation (different radiation type, doses and exposure times) and of the use of several parameters to describe plant growth with ambiguous definitions. Lethality is often used as the opposite of survival that is defined in various ways. More specifically, survival is described either as the percentage of plants with a minimum number of expanded leaves after an established number of days from sowing or as the number of plants that are able to reach the reproductive phase and can produce seeds (Zimmermann et al. 1996; Hase et al. 1999; Kumagai et al. 2000; Wu, and Yu 2001 and Shikazono et al. 2002). Whatever the definition, lethality and survival are much more complex concepts than germinability because they involve the further growth of viable plants with true leaves (Wareing and Phillips, 1981). The interpretation of results and the identification of common behaviours are complicated by the

75

variability of reactions found in different species and cultivars (Bayonove et al. 1984 and Yu et al. 2007). Also polyploidy reduces the radiosensitivity of plants; for example, there is evidence that, after high- LET irradiation, tetraploid lines of A. thaliana survive longer than diploid lines that are more easily subjected to the lack of germination or early death of seedlings (Gartenbach et al. 1996). As we explained in the previous section, polyploidy can hide recessive mutations (including the ones affecting the lifespan of the plant) due to the existence of supernumerary copies of the genome.

VIII- Haploid plants under Salinity stress The haploid plants of Nicotiana alata segments were cultured on MS medium supplemented with 0.2 mg IAA /l + 0.5 mg KIN /l and each containing one of different NaCl concentrations (0, 50, 100.150 or 200 mM /l). Table (9) showed that haploid plants were affected gradually by increasing the salinity levels, it's decreased to; 91.7, 79.2, 70.8 and 62.5 by increasing salinity, 50, 100, 150 and 200 mM NaCl, respectively. Fig (9) presented that the salinity effects of haploid plants survival and shoot length /cm were negatively. Whereas, the mortality percentage increased with increasing NaCl concentrations. Table (9) and Fig (9) determined that the highest survival percentage under salinity condition was 91.7% at 50 mM NaCl whereas the highest mortality percentage at 200 mM NaCl were reached to 62.5%. The salinity effects of haploid plants survival were significant (P≥ 0.76). Salinity is a major environmental constraint that renders fields unproductive and limits plant growth and productivity (Turkan and Demiral, 2009 and Khan et al., 2010). The adverse effects of salinity on plant growth are the result of changes in plant physiology which include ion toxicity, osmotic stress and nutrient deficiency (Munns and Tester, 2008 and Frary et al., 2010). However, Roshandel and Flowers (2009)

76

Table (9): Effect of NaCl concentrations on haploid plants buds survival and shoot length in Nicotiana alata after 40 days.

Haploid buds NaCl conc. survival Shoot length (mM.) Total % (cm) No.

0.0 100 96 100 7.2 50 100 88 91.7 6.6 100 100 76 79.2 5.3 150 100 68 70.8 4.6 200 100 60 62.5 3.9

LSD. (5%) 0.76

Control 50 100 150 200 mM mM mM mM

Fig. (9): Effect of NaCl concentrations on haploid plants survival in Nicotiana alata after 40 days.

77

have shown that the changes induced by salinity are ionic rather than osmotic.This conclusion is drawn from their molecular studies in which they found that the changes in the expression of genes encoding proline rich proteins, senescence associated proteins and heat-shock proteins were responsive to the ionic rather than osmotic effects of salt in rice. The toxic effect of salinity is through oxidative stress caused by enhanced production of reactive oxygen spices (ROS) (Zhu et al., 2007 and Giraud et al., 2008). The increased production of ROS occurs under all kinds of stress, although their identity and compartment of origin may differ (Li et al., 2009). The presence of high concentration of ROS can damage photosynthetic pigments, proteins, lipids and nucleic acids by oxidation (Halliwell and Gutteridge, 1985). Controlling ROS production and scavenging in the chloroplast is shown to be essential for tolerance to salinity in cabbage transgenic plants and in salinity tolerant cultivars (Tseng et al., 2007). In addition to the destructive role of ROS, they also serve as a stress signal molecule and activate acclimation and defense mechanisms that will in turn counteract stress-associated oxidative stress (Miller et al., 2008 & 2010; Frary et al., 2010). Recently, it has been shown that H2O2 produced by apoplastic polyamine oxidase influences the salinity stress signaling in tobacco and plays a role in balancing the plant response between stress tolerance and cell death (Moschou et al., 2008). Boursiac et al. (2008) have suggested a new role for ROS in regulating the gating of aquaporins in Arabidopsis roots during salt stress through a cellular signaling mechanism. Thus, ROS act both as the damaging toxic molecule and as the beneficial signal transduction molecule and there is need to control the steady-state level of ROS in cells during normal metabolism as well as in response to different stresses (Miller et al., 2010). Plants develop a plethora of biochemical and molecular mechanisms to cope with salt stress. Biochemical pathways leading to products and processes that improve salt tolerance are likely to act additively and probably synergistically (Iyengar and Reddy, 1996).High-complexity

78

mechanisms.Low-complexity mechanisms appear to involve changes in many biochemical pathways. High-complexity mechanisms involve changes that protect major processes such as photosynthesis and respiration, e.g., water use efficiency, and those that preserve such important features as cytoskeleton, cell wall, or plasma membrane–cell wall interactions (Botella et al., 1994) and chromosome and chromatin structure changes, i.e., DNA methylation, polyploidization, amplification of specific sequences, or DNA elimination (Walbot and Cullis, 1985). It is believed that for the protection of higher-order processes, low-complexity mechanisms are induced coordinately (Bohnert et al., 1995). Salt stress affects all the major processes such as growth, photosynthesis, protein synthesis, and energy and lipid metabolism. These are discussed under separate headings. Salinity stress results in a clear stunting of plants (Hernandez et al., 1995 and Cherian et al., 1999). The immediate response of salt stress is reduction in the rate of leaf surface expansion leading to cessation of expansion as salt concentration increases (Wang and Nil, 2000). Fresh and dry weights of plants increase with an increase in salinity in Salicornia rubra while the optimal growth occurrs at 200mM NaCl and the growth declines with a further increase in salinity (Khan, 2001). In Raphanus sativus (radish) total plant dry weight decreases at higher salinities and about 80% of the growth reduction at high salinity can be attributed to reduction of leaf area expansion and hence to reduction of light interception. The remaining 20% of the salinity effect on growth is most likely explained by a decrease in stomatal conductance. The small leaf area at high salinity is related to a reduced specific leaf area and increased tuber/shoot weight ratio and the latter can be attributed to tuber formation starting at a smaller plant size at high salinity (Marcelis and VanHooijdonk, 1999). Kurban et al. (1999) have reported that in Alhagi pseudoalhagi (a leguminous plant), total plant weight increases at low salinity (50 mM NaCl) but decreases at high salinity (100 and 200mM NaCl). Khan et al. (1999) have reported that when Halopyrum

79

mucronatum (aperennial grass found on the coastal dunes of Karachi, Pakistan) is treated with 0, 90, 180, and 360 mM NaCl in sand culture, fresh and dry mass of roots and shoots peaks at 90 mM NaCl, a further increase in salinity inhibits plant growth, ultimately resulting in plant death at 360 mM NaCl, and maximum succulence is noted at 90 mM NaCl. Experimental evidence shows that in a salt nonsecretor mangrove B. parviflora the plant growth is optimal at 100mM NaCl under hydroponic culture, whereas further increase in NaCl concentration retards plant growth and 500 mM NaCl is found to be lethal in this species (Parida et al., 2004). On the other hand a salt secretor mangrove Aegiceras corniculatum can tolerate up to 250mM NaCl and 300 mM is found lethal in this case (Mishra and Das, 2003). Increasing salinity is accompanied by significant reductions in shoot weight, plant height, number of leaves per plant, root length, and root surface area per plant in tomato (Mohammad et al., 1998). Increased NaCl levels results in a significant decrease in root, shoot, and leaf growth biomass and increase in root/shoot ratio in cotton (Meloni et al., 2001). IX- The gamma radiation and NaCl combined effect on haploid plantlets of Nicotiana alata. Nicotiana alata haploid plantlets cutted to segments after irradiated with doses 10, 15, 20 or 25 Gy and cultured on MS medium supplemented with 0.2 mg IAA /l + 0.5 mg KIN /l and containing defferent NaCl concentrations (50, 100,150 and 200 mM /l).Data obtained were presented in table (10) and Fig. (10). Data in table (10) showed that the combined effect of gamma radiation dose and salinity concentrations were negatively and gradually effected whereas the percentage of bud survivals and the plantlets lengths decreased with increasing gamma radiation dose and NaCl concentration ( Fig.10) regarding with the control (Figs; 8 & 9) . The lowest effect of the combined effect between gamma radiation doses and NaCl concentration were at 50 mM NaCl with rad. Dose 10 Gy. However, beginning of the 100 mM NaCl with rad. dose 25 Gy and 150 or 200 mM NaCl with rad. Doses more than 20 Gy were found to be lethal effect. 80

Table (10): The combined effect of gamma irradiation and salinity concentrations on 100 buds survival in Nicotiana alata.

Rad.dose (Gy) Na Cl con. ( mM) LSD. 0.0 50 100 150 200 (5%) Bud No. 96 88 76 68 60 % 100 91.7 79.2 70.8 62.5 0 Shoot lengh (cm) 7.2 6. 6 5.3 4.6 3.9 0.76

Bud No. 68 60 54 48 41 % 70.8 62.5 56 50 42.7 10 shoot lengh (cm) 4.6 4.3 4.0 3.8 3.2 0.39

Bud No. 49 44 40 36 32 % 51 45.8 41.6 37.5 33.3 15 shoot lengh (cm) 4.1 3.8 3.3 3.1 2.8 0.62

Bud No. 37 30 27 0 0 % 38 31 28 0 0 20 shoot lengh (cm) 3.6 3.0 2.6 0 0 0.34

Bud No. 29 21 0 0 0 % 30 21.8 0 0 0 25 shoot lengh (cm) 3.0 2.0 0 0 0 0.11

81

10 Gy X 50 10 Gy X 100 10 Gy X 150 10 Gy X 200 mM mM mM mM

15Gy x 50 15Gy x 100 15Gy x 150 15Gy x 200 mM mM mM mM

Fig. (10): combined effect of gamma irradiation and NaCl concentration on haploid buds survival in Nicotiana alata

82

Fig. (10) cont.

Control 20Gyx 50 20 Gyx 100 20 Gy m.mol m.mol

Control 25 Gyx 50 25 Gy m.mol

Control 25 Gyx 50 25 Gy m.mol

83

The effect of gamma radiation doses (0.0, 10, 15, 20 and 25 Gy) and NaCl concentrations (0.0, 50, 100, 150 and 200 mM /l) on Nicotiana alata anther survival and shoot length were significant (P ≥ 0.76, 0.39, 0.62, 0.34 and 0.11; respectively) Salinity stress affects many metabolic aspects and induces anatomical and morphological changes resulting in reducing growth. These results are in line with Mostafa (2002) who found that the combined effects between gamma radiation and NaCl on callus growth rate, buds survival and shoot length decreased by increasing gamma radiation doses and NaCl concentrations. Concerning the effect of radiation, it is clear from table (10) and Fig. (10) that the values of survival percentage, and the length were of shoots decreased significantly by increasing gamma irradiation doses (10 and 40 Gy) compared with control (Eliwa 2001 and 2008) on banana cultivars which was found the same results, and also agreement with El Sayed, (2006) who found that the combined effects between gamma radiation doses and NaCl concentrations on callus growth rate and differentiation of Catharanthus sp. were decreased by increasing gamma radiation doses, NaCl concentrations or both treatments.

84

X- The proline contents; Biochemical Analysis in haploid plants a-Under Radiation stress: Proline contents were estimated in haploidplants with and without irradiate stress controlled with diploid plants Proline content. Data in Table (11) showed that proline contents in Nicotiana alata diploid plants were (0.37 mg / 100 g fresh wt) whereas the haploid plants were (0.18 mg / 100 g fresh wt). Proline contents in the haploid plants irradiated with doses (2.5, 5, 7.5,10, 15, 20 and 25 Gy) were increased with increasing gamma radiation doses (1.09, 1.13, 1.23, 1.25, 1.34, 1.43and 1.50 mg/100g fresh wt respectively).The use of dose 25 Gy induced the maximum production of proline content in Nicotiana alata haploid plants to 1.50 mg/100g fresh wt. The haploid proline production was found to be half of diploid proline production. The proline production was increased with increasing gamma radiation doses.

Table (11): Effect of gamma radiation on proline content (mg/100g fresh wt.) in Nicotiana alata

plants Radiation dose Proline (Gy) (mg) Diploid 0.0 0.37 Haploid 0.0 0.18 Haploid 2.5 1.09 Haploid 5 1.13 Haploid 7.5 1.23 Haploid 10 1.25 Haploid 15 1.34 Haploid 20 1.43 Haploid 25 1.50

85 b-Under salt stress: The proline contents were evaluated in Nicotiana alata haploid plants were grown under saline conditions .Haploid plants were cultured on MS medium supplemented with 0.2 mg IAA+0.5 mg KIN /l. Results obtained were presented in table (12).This table illustrated that the different NaCl concentrations (0.0, 50, 100, 150 and 200 mM) induced a highly increasing in haploid proline content to (1.55, 1.61, 1.71 and 1.88 mg / 100 g fresh wt. respectively). Finally, Salt stress was induced a sudden increasing in Proline content in haploid Nicotiana alata plants,

Table (12): Effect of NaCl on Proline content (mg/100g fresh wt.) in Nicotiana alata haploid plants.

plants NaCl Conc. Proline (mM ) (mg) Diploid 0.0 0.37 Haploid 0.0 0.18

Haploid 50 1.55 Haploid 100 1.61 Haploid 150 1.71

Haploid 200 1.88

Radiation caused oxidative injury by accelerating free radical generation in living systems. The primary damage induced by ionizing radiation is modified in enzymatic repair processes Alikamanoglu et al. (2007). Proline is one of the most common compatible solutes accumulated in stressed plants and acts as a non-toxic osmotic solute and may supply energy to increase salinity tolerance (Franco et al.1999).They added that proline is osmotic adjustment, preserves enzymes and other important

86 cellular structures, is assumed to scavenge hydroxyl radicals and is a reserve for carbon and nitrogen immediately after the relief of stress. Many studies on proline synthesis and catabolism genes have provided results that are consistent with diverse functions of proline as a source of energy, nitrogen and carbon, and as an osmolyte in response to dehydration Peng et al., (1996).The proline increased with increasing gamma radiation doses. This agrees with Mostafa (2002) who found that the doses (10, 20, 30 and 40 Gy) increased proline content in sweet potato for cvs. Mabruka and Abees. However, doses (50, 60, 70 and 80 Gy) decreased this trait. Eliwa, (2001) reported that proline concentration increased significantly in both banana cvs. (Williams & Grand Nain) as the irradiation doses increased up to 40 Gy but at 60, Gy reverse is true. Proline concentration in the selected cells rose steeply with external NaCl, particularly so above 100 millimolar NaCl. The NaCl selected cells responded to water stress (i.e., added mannitol) by accumulating markedly more proline than did the wild type (Wattad et al., 1983). Accumulation of proline in salinized banana shoots can be explained by the higher inhibitory rate of proline dehydrogenase and proline oxidase. Thus, accumulation of proline oxidation or diminished incorporation of proline into protein due to impaired protein synthesis and reduced growth. Proline protects protein structures against denaturation and protects cell membrane to prevailing environmental stress conditions like salinity (Mansour, 1998). The increased proline utilization suggests increasing the incorporation of proline into synthesis of cell wall proteins. This might be involved in morphological change, which is one of the well-known responses under salt stress (Ueda et al., 2007). Moreover, salt stress leads to changes in gene expression, leading to an increased synthesis of osmoprotectors and osmoregulators (Hong-Bo et al., 2006). Osmotic adjustment is the main component of physiological machinery by which plants respond to soil-salt stress. The accumulation of proline and soluble sugars in stressed plants is very important for plant adaptation during stress

87 (Tan et al., 2006) on Radix astragali; (Manivannan et al., 2007) on Vigna radiata and Jaleel et al,. (2007) on Catharanthus roseus. One of the explanations of this large success is that this resistance in most cases not from gene mutation but from physiological adaptation which may hinder the selection of salt resistant mutant cells. Proline acts as an enzyme protectant, stabilizing the structure of macromolecules in pea nut and as a major reservoir of energy and nitrogen for utilizing upon exposure to salinity (Girija et al., 2002). The increased proline levels are another common response of plants upon osmotic stress (Karabal et al., 2003). Proline has a function of osmotic adjustment in plants, but it also protects enzymes and membranes against oxidative stress in Cassia angutifolia (Claussen, 2005). Agarwal and Pandey (2004) concluded that proline is a reliable indicator of the environmental stress imposed on plants, thus allowing us to establish stress thresholds for fruit yield and product quality of tomato. Osmotic adjustment through the accumulation of cellular solutes, such as proline has been suggested as one of the possible means for overcoming osmotic stress caused by the loss of water (Shankhadhar et al., 2000). Free proline accumulation increased many folds upon exposure to abiotic stresses (Ahmad et al., 2006 and 2007). Proline has been shown to accumulate in plant cells exposed to stress conditions (Joyce et al., 1992). In nature, drought resistant and salt tolerance is often found in the same plant, since both traits are useful in typical arid environments. There are some circumstantial indications that NaCl tolerant plants are also drought resistant (Thinh, 1995).The results here agree with (Mostafa, 2002 and Locy, 1995). This indicates that uptake and metabolism of N is so triggered under abiotic stress that specific soluble nitrogenous compounds accumulated which provide adaptive value to the plants. In majority of plants, abiotic stress leads to changes in gene expression, leading to an increased synthesis of osmoprotectors and osmoregulators. Free proline accumulation in the leaves under stress conditions are important for plant adaptation during stress (Tan et al., 2006). In most plants, there is an increased accumulation of

88 amino acids and amines (e.g. proline, B-alanine, glycine betain) in their tissues in response to water deficit. Generally, plant species that accumulate proline usually have low amounts of this amino acid when grown in well-watered and non-saline soils, increasing its contents upon imposition of drought or waterdeficits on potato (Teixeira and Pereira, 2007). Under abiotic stress, total amino acids decreased in shoots of 3 weeks old plants, but increased significantly in 5 weeks old plants. However proline and proline amino acid ratio levels progressively increased in shoots of 3and 5 weeks old maize plants (Kerrit, 2005). Many studies on proline synthesis and catabolism genes have provided results that are consistent with diverse functions of proline as a source of energy, nitrogen and carbon, and as an osmolyte in response to dehydration ( Peng et al., 1996). c- The combined effects between gamma radiation doses and NaCl concentrations on proline contents in haploid N. alata plants. proline content in Nicotiana alata haploid plants which exposed to gamma radiation doses (10, 15, 20 and 25 Gy) and grown on MS medium containing one of each different NaCl concentrations (0,50, 100, 150 or 200 mM) were estimated and presented in table 18. Data in table (13) were clearly indicate that the proline contents were decreasedby increasing either radiation dose or Na Cl concentration compared with the control. However, starting from the cmbined effects between dose 25Gy with NaCl more than 50mM, the growth of plants were failed,also the same was found on more than 100 mM Na Cl with 20 Gy gamma rad.Generally, The proline contents were decreased with the combined effects between gamma radiation doses and salinity compaining with control, it was stable and gave about half of the control.

89 Table (13): The combined effects between gamma radiation doses and NaCl concentrations on proline contents (mg/100g fresh wt.) in Nicotiana alata haploid plants.

NaCl

conc. Radiation dose ( Gy.) mM 0 10 15 20 25

0.0 0.18 1.25 1.34 1.43 1.50 50 1.55 0.55 0.89 0.99 1.05 100 1.61 0.71 0.95 1.06 0 150 1.71 0.77 0.99 0 0 200 1.88 0.83 1.08 0 0

The accumulation of compatible solutes is often regarded as a basic strategy for the production and survival of plants under abiotic stress condition, including both salinity and oxidative stress (Chen et al., 2007). Eliwa (2001) found that proline content increased significantly in both banana cultivars (Williams and Grand Nain) by gamma irradiation doses (20 & 40 Gy ) while, the interaction between gamma irradiation and salinity caused an increase in proline content at low doses. High doses of gamma radiation and salt concentration caused a sharp decrease in proline content. Aly (2000) found an increase in the accumulation of proline in both minitubers and macrotubers of potato cv. Diamant with increasing salt concentration and gamma irradiation dose as well as the interaction between them. As an interaction of these effects, the water potential of cells may be decreased by the synthesis and accumulation of compatible osmolytes such as proline, glycin betaine and sugar allowing additional water to be taken up. Abiotic stress resulted in an increase of proline biosynthesis rate (Rhodes and Handa, 1989). Proline is often considered as a compatible solute involved in osmotic adjustment (Azooz et al., 2004 & Kavikishore et al.,

90 2005). Proline may act as a osmoregulator compound to equilibrate the osmotic potential in bean cell (Cárdenas-Avila et al., 2006). Proline accumulation may in part involve induction (Peng et al., 1996) and / or activation of enzymes of proline feedback inhibition control of the pathway (Delauney and Verma, 1993), decreased proline oxidation to glutamate (Elthon and Stewart, 1982) mediated at least in part by down –regulation of proline dehydrogenase (Kiyosue et al., 1996 and Peng et al., 1996), decrease utilization of proline in protein synthesis (Stewart, 1981) and enhanced protein turnover (Fukutoku and Yamada, 1984).

B- Total protein a) Under radiation stress The irradiated haploid and diploid Nicotiana alata plants with doses (0, 2.5, 5, 7.5, 10, 15, 20 or 25 Gy) were estimated in table (14). The total protein in diploid plants were gave a two folds more than haploid plants, while its increased in haploid plants with increasing gamma radiation doses, the maximum protein production was found by dose 10 Gy (2.95 mg / 100 g fresh wt)

Table (14): Effect of gamma radiation on total protein content (mg/100g fresh wt.) in Nicotiana alata.

plants Radiation dose Total protein (Gy) (mg) Diploid 0.0 1.41 Haploid 0.0 0.98 Haploid 2.5 1.72 Haploid 5 1.98

Haploid 7.5 2.16 Haploid 10 2.95 Haploid 15 2.14 Haploid 20 1.76

Haploid 25 0.82

91 b-Under salt stress

Nicotiana alata diploid and haploid plants were cultured on MS medium supplemented with 0.2 IAA mg/l + 0.5 KIN mg/l and containing one of each of different NaCl concentrations(0, 50, 100 ,150 or 200 NaCl mM) were estimated and presented in Table 15. The total protein was increased with increasing NaCl concentrations. Also, the total protein in diploid plants was about 1.5 fold more than the haploid plants.

Table (15): Effect of salinity on total protein (mg/100g fresh wt.) in Nicotiana alata.

Plants NaCl conc. Total (mM) protein

(mg) Diploid 0.0 1.41 Haploid 0.0 0.98 Haploid 50 1.13

Haploid 100 1.65 Haploid 150 2.11 Haploid 200 2.24

It was previously shown that gamma irradiation significantly influences the cell metabolism and protein synthesis in plant meristem cells (Casarett, 1968) According to the results obtained in the present study, it was observed that increased gamma dosage caused a reduction of total soluble protein content. However, plants irradiated at relatively low dosage (10 and 20Gy) displayed a higher total soluble protein content compared to their non- irradiated counterparts. This result demonstrated that there was a direct correlation between gamma dosage and protein content.

92 Gamma irradiation caused inhibition of tissue culture growth along with failure of RNA, and subsequently of protein synthesis (Bajaj, 1970). This accounts for the lower protein concentration in plants irradiated at high dosage (70Gy). The most crucial function of plant cell is to respond to gamma stress by developing defense mechanisms. This defense was brought by alteration in the pattern of gene expression (Corthals et al., 2000). This led to modulation of certain metabolic and defensive pathways (Zolla et al., 2003). Owing to gene expression altered under gamma stress, qualitative and quantitative changes in total soluble protein contents were obvious (Corthals et al., 2000). These proteins might play a role in signal transduction, antioxidative defense, antifreezing, heat shock, metal binding, antipathogenesis or osmolyte synthesis which were essential to a plant’s function and growth (Gygi et al., 1999) In a study by Bajaj (1970) which analyzed the effect of gamma irradiation on soluble protein content of bean callus culture, it had been reported that at high irradiation dosage (80Gy), soluble protein content continue to decrease. According to the results obtained in the present study, it was observed that increased gamma dosage caused a reduction of total soluble protein content. However, plants irradiated at relatively low dosage (10 and 20Gy) displayed a higher total soluble protein content compared to their non-irradiated counterparts. This result demonstrated that there was a direct correlation between gamma dosage and protein content. According to Bajaj (1970), gamma irradiation caused inhibition of tissue culture growth along with failure of RNA and subsequently the failure of protein synthesis. In accordance with the results obtained by Stajner et al. (2007) in the study of soybean seeds, 10Gy dosage caused a slight increased in total soluble protein content, an increase of 11.0% as compared to the non irradiated seeds. Data obtained by Cho and Song (2000) showed that gamma irradiation, did not induce significant loss in water soluble components such as total soluble proteins, minerals, nitrogenous constituents, and sugars. Stajner et al. (2007) also revealed that at an increased gamma dosage, the quantity of carbonyl groups in oxidatively modified proteins significantly increased. Introduction of carbonyl groups into amino acid residues of proteins was a

93 hallmark for oxidative modification due to gamma rays exposure. Gamma irradiation creates oxidative stress and affects biomolecules by causing conformational changes, oxidation, rupture of covalent bonds and formation of free radicals such as the hydroxyl and superoxide anion that were generated by radiation (Variyar et al., 2004). These free radicals could modify the molecular properties of the total soluble proteins causing oxidative modifications of the proteins (Wilkinson and Gould, 1996). The chemical changes caused by gamma irradiation in proteins were fragmentation, cross-linking, aggregation, and oxidation by oxygen radicals generated in the radiolysis of water (Davies and Delsignore, 1987). These changes depend on the chemical nature of the protein, its physical state, and the irradiation condition (Woods and Pickaev, 1994). Especially, the effect of gamma irradiation on protein conformation appears to depend on several factors such as protein concentration, the presence of oxygen, and the quaternary structure of proteins (Garrison, 1987). In general, radiation causes the irreversible changes of protein conformation at the molecular level by breakage of covalent bonds of polypeptide chains (Kume and Matsuda, 1995). Fragmentation involves reaction of a-carbon radicals with oxygen to form peroxyl radicals which decompose to fragment the polypeptide chain at the a- carbon. Hydroxy radical and superoxide anion radical generated by radiation could modify primary structure of proteins, which resulted in changes of molecular weight distribution (Garrison, 1987). Besides fragmentation, aggregation of proteins fragmented is also observed. There have been reports on aggregation and cross- linking of proteins by irradiation (Filali-Mouhim et al., 1997). Covalent cross-linkages are formed between soluble proteins and between peptides and proteins (Garrison, 1987). Grechenkov and Serebrenkov 1962) found that total free amino acids, protein synthesis and accumulation increased in gamma irradiated potato plants. However, (Tikhonov et al. 1980) reported that the irradiation of Datura innoxia seeds by gamma rays at 0.5 - 1.0 Krad reduced total protein content. Shahine et al. (1988) found that the exposure grains of Giza barely to gamma rays by dose of 2 Krad increased protein content and reached to its maximum values

94 under the influence of this dosage. Hammam (1992) studied the effect of low doses of gamma radiation (4, 8, 12 and 16 Krad) as pre-sowing treatment on protein and free amino acids content of sunflower seeds. He found that irradiation dose of 12 and 14 Krad decreased total protein content. All applied doses of gamma radiation increased the free amino acids content. But, Sakr (1992) stated that the protein content of leaves, stems and flowers increased as a result of irradiating Hyoscyamus muticus L. with gamma rays at 2, 4, 8, 16, 24 and 32 Krad. He added also that except those of 24 and 32 Krad doses that caused decreases in total protein content in leaves. also 16 and 24 Krad doses caused significant increase in protein content of stem. Selim and Atia (1996) found that protein concentration of wheat grains was increased by increasing gamma doses up to 10 Krad, and then decreased by doses of 15 up to 30 Krad. Moustafa (2002) found that the use of different doses from gamma radiation induced changing in total protein in both cvs. Mabruka and Abees. The effect of gamma radiation doses in cv. Mabruka on total protein were increased, but the increasing in total protein were variable. While the same doses effects in total protein in cv. Abees were fluctuate, some doses induced increasing like 10, 40, 70 and 80 Gy and the other doses (20, 30, 50 and 60 Gy) caused decreasing in total protein. Generally, the total protein content was a high in cv. Mabruka to more than cv. Abees. c- The combined effects of gamma radiation and salinity on total protein The combined effects between gamma radiation doses (10, 15, 20 and 25 Gy and NaCl concentrations (50, 100, 150 and 200 ml) on total protein in plantlets were estimated. The combined effects between gamma radiation dose and saline levels on total protein in Nicotiana alata haploid plantlets were cultured on MS medium supplemented with 0.2 mg IAA + 0.5 mg KIN /l were also studied (table 16).The irradiated plantlets with dose 10 Gy caused increasing in total protein at (50 100, 150 and 200 ml) NaCl to (1.81, 1.99, 2.17 and 2.12 mg/100 fresh wt. respectively). The effect of dose (15 Gy) induced decreasing at all

95 salinity levels (50, 100, 150 and 200 ml) to (0.89, 0.77, 0.62 and 0.56 mg/100 fresh wt. respectively). The use of dose 20 Gy induced decreasing in total protein with NaCl concentrations (50 and 100 ml) to (1.92 and 1.68 mg/100 fresh wt. respectively). Dose 25 Gy induced decreasing in total protein at saline concentrations 50 ml NaCl to (1.14mg/100 fresh wt.) The conclusion form data in tables (14 and 15) showed that gamma radiation doses (0.0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy) and saline concentrations (0.0, 50, 100, 150 and 200 ml) induced increasing in total protein. The combined effects between gamma radiation doses (10, 15, 20, and 25 Gy) and NaCl concentrations (50, 100, 150 and 200 ml) on total protein were decreased (table, 16)

Table (16): The combined effects of gamma radiation doses and NaCl concentrations on total protein (mg/100 gm fresh wt.) in Nicotiana alata haploid plants.

NaCl Radiation dose/Gy. conc. 20 mM 0 10 15 25

0.0 0.98 2.95 2.14 1.76 0.82 50 1.13 1.81 0.89 1.92 1.14 100 1.65 1.99 0.77 1.68 0.0

150 2.11 2.17 0.62 0.0 0.0 200 2.24 2.12 0.56 0.0 0.0

Results obtained are inagreement with Aly (2000) who studied the change in total crude protein of potato tubers and macrotubers of the two cultivars Cara and Diamont resulted from salt stress and gamma irradiation treatments. Salt stress 2000 ppm and 4000 ppm NaCl caused slightly increases in the minitubers and macrotubers of potato cultivar and reached the maximum at salt level 4000 ppm NaCl to 11.5% and 12.9% D.W. for minitubers and macrotubers of potato cv. Cara and Diamont; respectively.Protein

96 are known to be induced by salt stress (Close et al., 1989 in barely and Kijosue et al., 1994 in Arabidopsis thaliana). Abiotic stress leads to changes in gene expression, leading to an increased synthesis of osmoprotectors and osmoregulators. The enhanced tolerance to oxidative stress, brought about by constitutive expression of a single protein might result from a number of different mechanisms: (1) the expressed protein might have a direct scavenging activity that can detoxify certain species of reactive oxygen; (2) the expressed protein might be a part of a cellular network, or signal transduction pathway that protects the cell from damage caused by oxidative stress or is involved in the detoxification of reactive oxygen, and its constitutive expression enhances the activity of this network or pathway; or (3) the expressed protein might alter plant metabolism, causing the accumulation of reactive oxygen and indirectly activating the cells' scavenging, protection, and/or repair mechanisms against oxidative stress similar to some transgene-induced lesion mimics. When cells are exposed to elevated temperature or other environmental stresses, heat shock proteins belonging to several gene families are induced. Many heat shock protein function as chaperones and plays important roles in normal growth and stress tolerance. There is increasing evidence that the function of chaperones in preventing or reversing protein aggregation is linked to their role presenting misfolded intermediated to the cellular machinery proteolytic degradation (Hayes and Dice, 1996).

97 C-Molecular Markers 1- SDS- PAGE electrophoresis 1-The effect of ploidy level on protein bands; Protein extracts of haploid and diploid plantlets of Nicotiana alata were subjected to analysis by SDS-PAGE. Table (17) and Figure (11) showed that the changes in protein banding patterns were studied in diploid and haploid plantlets. The numbers of protein bands in diploid plantlets are (10) but in haploid plantlets are (8). The protein bands in diploid plantlets with molecular weight (109., 91, 73, 63, 44, 41, 37, 34, 29 and 25 KDa, respectively) were appeared with band intensity (11. 083, 14.849, 21.765, 10.14, 2.851, 2.252, 6.935, 13.36, 6.604and 22.491 % respectively). The 109 KDa band was appeared as a genetic marker of diploid plantes. The haploid plantlets have one new band with molecular weight (31KDa) with band intensity (24.41%). Three bands with molecular weight (109, 91 and 73KDa were disappeared. 2- Effect of gamma radiation doses on SDS-PAGE banding pattern of Nicotiana alata plantlets: The protein pattern of cultured haploid Nicotiana alata on MS medium supplemented with 0.2 mg IAA + 0.5 mg KIN/l and exposed to gamma radiation doses (0, 2.5, 5.7, 5, 10, 15, 20 and 25 Gy) were investigated. Data in Table (17) and figure (11) were also showed clearly that the effect of gamma radiation dose 2.5 Gy on protein banding patterns caused decrease to seven bands of total protein banding. One band with molecular weights (49KDa) with band intensity (1.943 %) was appeared. Two bands with molecular weights 44 and 37KDa respectively) were disappeared. The dose 5 Gy caused an increase by two bands more than haploid in number of protein banding (from 8 to 10). Four new bands were appeared with molecular weights 114, 91, 73 and 56 KDa with protein band intensity (3.723, 3.93, 2.213 and 8.753 % respectively). Two bands with molecular weights (63 and 44 KDa were disappeared. The 56 KDa band was appered as agenetic marker of this dose. The total numbers of protein bands were increased by one band (from 8 to 9) with dose 7.5 Gy controlled with haploid plantlets. Three new

98 bands with molecular weights (91, 73 and 61 KDa,) with protein band intensity (6.306, 8.051 and 8.108 % respectively) were appeared. While two bands with molecular weights 63 and 44 KDa were disappeared. The gamma radiation dose 10 Gy caused decreasing one band loss than haploid in total number of protein bands (from 8 to 7). Two new bands with molecular weights (119.639 and 60.927 KDa respectively) with band intensity (3.97and 4.917 % respectively) were appeared. Two bands with molecular weights (63 and 44 KDa) were disappeared. The effect of gamma radiation dose 15 Gy on protein bands induced appearing of two new bands with molecular weights; (114 and 61 KDa) with protein band intensity; 4.592 and 6.397 %; respectively while two other bands with molecular weights 63 and 44 KDa were disappeared controlled with haploid plantes. The use of dose 20 Gy induced increasing one band in number of protein bands (from 8 to 9) more than haploid. Two new bands with molecular weights 118 and 49 KDa with protein band intensity (6.321 and 3.844 % respectively) were appeared. One band with molecular weights44 KDa was disappeared. 25 Gy radiation dose was induced a three new bands controlled with haploid plantlets with molecular weights 105, 73 and 61 KDa with protein band intensity; 10.284, 9.828 and 8.643 %; respectively. Two bands with molecular weights (63 and 44 KDa ) were disappeared. A unique band was appeared to be a genetic marker for this dose (105 KD). Finely the gamma radiation doses (0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy) induced qualitative and quantitative differences represented in appearance of new bands, disappearance of some bands and changes in band intensity.

99 Table (17): Comparative analysis of the effect of gamma radiation doses on molecular weight and protein intensity (I) in Nicotiana alata plantlets by using SDS-PAGE technique.

Radiation Dose / Gy Cont Cont 2.5 5 7.5 10 15 20 25 M.W. of dip hap Band I I I I I I I I I

118 3.97 6.321

114 3.723 4.592

109 11.083

105 10.284

91 14.849 3.93 6.306

73 21.765 2.213 8.051 9.828

63 10.14 13.30 8.77 12.962

61 8.108 4.917 6.397 8.643

56 8.753

49 1.943 3.844

44 2.851 4.46

41 2.252 4.28 2.983 3.786 6.265 5.801 4.657

37 6.935 4.04 5.967 2.58 53.642 4.523 5.782 6.324

34 13.36 32.04 18.995 26.408 30.245 52.409 18.283 18.545 67.107

31 24.41 14.199 91.413 23.064 16.527 25.566 22.223 18.336

29 6.604 10.80 5.195 2.132 6.632 4.628 6.781 11.903 7.436

25 22.491 68.99 50.01 29.328 47.377 66.73 76.659 58.093 61.535

Total .No. 10 8 7 10 9 7 8 9 8 of band

100

Fig (11): Gamma rays effect on SDS –PAGE of protein electrophoresis gel of Nicotiana alata

101 3-Effect of NaCl concentrations on protein banding patterns in Nicotiana alata plantlets: The changes in protein banding patterns in haploid Nicotiana alata plantlets adapted to a medium MS + 0.2 mg IAA + 0.5 mg KIN/l and containing one of each, NaCl concentrations; 50, 100, 150 and 200 mM and controlled with diploid and haploid plantlets with 0.0 mM NaCl were studied. Table (18) and figure (12) represent the salt effects on banding patterns in Nicotiana alata plantlets. The number of protein bands in haploid (8) was affected with salt concentrations 50, 100, 150 and 200 mM becomes 8, 10, 9, and 9; respectively). The effect of 50 mM NaCl on protein bands induced appeared two new band with molecular weight (61 and 49 KDa) with protein band intensity 5.816 and 10.01 %; respectively. Three bands with molecular weights (63, 44 and 37 KDa) were disappeared.Band of 61 KDa was found to be a unqie band in this dose. The concentration 100 mM NaCl induced three new bands with molecular weights; (114, 91 and 56 KDa) with band intensity; 19.509, 17.115 and 29.646 % respectively. One band with molecular weight (63 KDa) was disappeared.The 114 KDa band was appeared as a genetic marker of this concentration. The plantlets grown on 150 mM salt concentration; caused appearance two new bands with molecular weights (109 and 56 KDa) with protein band intensity 4.462 and 16.366 %; respectively. One band with molecular weight 63 KDa was disappeared. The concentration of 200 mM NaCl caused the presence of three new bands with molecular weights (49, 22 and 20 KDa) with band intensity 7.371, 10.446 and 34.732 %; respectively. Two bands with molecular weights (63 and 44 KDa) were disappeared controlled with the haploid plantlets. Bands with 22& 20 KDa were found as genetic marker for this concentration.

102 Table (18): Comparative analysis of salinity on molecular weight and protein band intensity (I) in Nicotiana alata plantlets using SDS- PAGE.

NaCl concentration (mM) M.W. of Cont.dip. Cont.hap. 50 100 150 200 Band I i I I I I

118

114 19.509

109 11.083 110.054

105

91 14.849 17.115

73 21.765

63 10.14 13.30

61 5.816

56 29.646 57.897

49 10.01 7.371

44 2.851 4.46 5.91 44.783

41 2.252 4.28 6.362 7.605 41.153 5.287

37 6.935 4.04 12.099 20.587 37.981 15.821

34 13.36 32.04 47.712 71.334 33.378 38.086

31 24.41 26.98 18.446 31.052 47.53

29 6.604 10.80 8.904 6.883 28.691 7.25

25 22.491 68.99 26.322 126.77 24.926 4.853

22 10.446

20 34.732

Total .No. 10 8 8 10 9 9 of band

103

Fig (12): Na Cl effect on SDS –PAGE of protein electrophoresis of Nicotiana alata.

104 4) The combined effects between gamma radiation doses and salinity on protein banding pattern. The SDS-PAGE banding pattern of irradiated haploid plantlets of Nicotiana alata with doses (10, 15, 20 and 25 Gy) grew on MS medium + 0.2 mg IAA + 0.5 mg KIN/l and supplemented with different concentrations of 50, 100, 150 and 200 mM NaCl were studied. Data in Table (19) and figure (13) determined that the combined effect of gamma radiation dose 10 Gy and NaCl concentrations (50, 100, 150 and 200 ml mol) on the changes of protein banding patterns. The combined effects of dose 10 Gy and 50 ml mol NaCl changes in appearing three new bands with molecular weights (49, 27and 22KDa) with band intensity 4.643, 10.046 and 8.065 %; respectively . Also three bands with molecular weights 63, 44 and 41 KDa were disappeared. The effect of dose 10 Gy and 100 mM salt concentration induced the appearing three new protein bands with molecular weights (49, 27 and 22KDa), respectively with protein band intensity 8.615, 10.979 and 9.326 %; respectively. Four bands with molecular weights (63, 44 and 41 KDa) were disappeared. The use of dose 10 Gy and 150 mM resulted in appearance of three new bands with molecular weights (49, 27and 22 KDa) with band intensity 3.429, 5.689 and 23.078 %; respectively. Four bands with molecular weights; 63, 44, 41and 37KDa were disappeared. The total number of protein band decrease one band (from 8 to 7). The combined effect of gamma radiation dose 10 Gy and 200 mM NaCl concentration induced two new bands with molecular weights (56 and 27KDa) appeared with protein intensity (5.571 & 4.041 %; respectively. Three bands with molecular weights 63, 44 and 22 KDa; respectively were disappeared. The companied effects between dose 10 Gy and (50, 100, 150 and 200 mM NaCl concentrations) induced quantitative and qualitative changes. These changes observed in appearing new bands, also

105 Table (19): Comparative analysis of the combined effect of gamma radiation dose 10 Gy and salinity on molecular weight and protein band intensity (I) in Nicotiana alata plantlets using SDS –PAGE.

NaCl concentration (mM) M.W. of Cont.dip. Cont.hap. 10 × 50 10×100 10 × 150 10× 200 Band I I I I I I

118

114 109 11.083 105

91 14.849

73 21.765 63 10.14 13.30 61 8.615

56 5.571

49 4.643 6.892 3.429 44 2.851 4.46 41 2.252 4.28 21.94 7.046

37 6.935 4.04 9.155 32.936 16.46

34 13.36 32.04 18.028 27.187 11.519 27.121 31 24.41 36.906 16.602 39.085 29 6.604 10.80 28.445 10.979 12.949 15.314

27 10.046 14.049 5.689 4.041

25 22.491 68.99 14.59 8.496 21.45 22 8.065 9.326 23.078

Total .No. 10 8 8 8 7 8 of band

106

250

Fig (13): Combined effect of gamma rays (10Gy) and salinity on SDS –PAGE of protein electrophoresis of Nicotiana alata

107 disappearing some bands and in band intensity. There were two bands with molecular weights (63and 44 KDa ) were disappeared and appearance new three bands with molecular weight (49, 27 and 22 KDa) detected in Nicotiana alata plantlets in these treatments. The band of 56 KDa was appeared only in 200 mM NaCl as shown in table (19).

Table (20) and figures (13&14) showed that the combined effects of radiation dose 15 Gy and salt concentrations; 50, 100, 150 and 200 mM on protein banding patterns in Nicotiana alata plantlets. The number of protein bands increased from 8(haploid) to; 9, 9, 10 and 9 with treatments 50, 100, 150 and 200, respectively.The use of dose 15 Gy and 50 mM NaCl concentration induced four new protein bands with molecular weights 56 and 20 KDa with band intensity (2.62 and 17.688 %; respectively) were appeared. One band with molecular weights 63 KDa was disappeared. The effects of gamma radiation dose 15 Gy and 100 ml mol salt concentration induced changes in protein bands. Three new bands with molecular weights 114, 91and 20 KDa with band intensity (3.196, 0.481and 9.643 %, respectively) were appeared. Two bands with molecular weights (63 and 25KDa) were disappeared. Six new bands were appeared with dose 15 Gy and 150 mM NaCl with molecular weight; 109, 100, 91, 63, 49 and 20 KDa with band intensity (2.309, 2.252, 2.623, 2.555, 3.351 and 8.004 %, respectively). Four bands with molecular weight 63, 41, 37, and 25 KDa were disappeared. The interaction between 15 Gy and 200 mM induced two new bands with molecular weight 56 and 20KDa with band intensity; 3.548and 22.06 %, respectively. One band was disappeared with molecular weight 63 KDa. The combined effects of gamma radiation dose 15 Gy and NaCl concentrations (50, 100, 150 and 200 mM) induced qualitative and quantitative changes. These changes observed in appearance new bands, disappearance some bands and protein band intensity. There were seven new bands appeared with molecular weight 114, 109, 100, 91, 63, 61 and 56 as a selective markers in Nicotiana alata plantlets irradiated with 15 Gy and grew on salt concentrations (50, 100, 150 and 200 mM).

108 Table (20):Comparative analysis of the combined effect of gamma radiation dose 15 Gy and salinity on molecular weight and protein band intensity (I) in Nicotiana alata plantlets using SDS –PAGE.

NaCl concentration (mM) M.W. of Cont.dip. Cont.hap. 15 × 50 15×100 15 × 150 15× 200 Band I I I I I I

118

114 3.196

109 11.083 2.309

100 2.252

105

91 14.849 0.481 2.623

73 21.765

63 10.14 13.30 2.555

61 9.643

56 2.62 55.439

49 3.351

44 2.851 4.46 1.643 4.351 5.137 2.479

41 2.252 4.28 10.614 3.212 4.531

37 6.935 4.04 31.198 4.027 5.754

34 13.36 32.04 14.794 16.113 26.351 18.60

31 24.41 5.857 13.651 10.627 11.449

29 6.604 10.80 9.617 7.887 5.068

25 22.491 68.99 5.028 6.163 5.738

20 17.688 8.004 22.06

Total .No. 10 8 9 9 10 9 of band

109

Fig (14): SDS –PAGE of protein electrophoresis gel of Nicotiana alata combined effect between gamma radiation doses (15,20and25 Gy) and salt concentrations (0, 50, 100, 150 and 200 mM).

110 The irradiated Nicotiana alata with dose 20 Gy and adapted in media (MS + 0.2 mg IAA + 0.5 mg KIN/l) and supplemented with different concentrations of NaCl (50, 100, 150 and 200 mM ) induced increasing in the number of protein bands ( from 8 to 9). Data in Table (21) represent the combined effects between gamma radiation dose 20 Gy and salt concentrations (50, 100, 150 and 200 mM) NaCl on protein banding patterns in Nicotiana alata plantlets. The dose 20 Gy and 50 mM NaCl concentration due to appeared five new protein bands with molecular weights; 109, 85, 56, 49 and 21KDa with band intensity; 4.519, 4.443, 4.368, 1.665 and 23.612 %; respectively. Four bands with molecular weights (63, 44, 34 and 31 KDa respectively were disappeared. The same dose 20 Gy with 100 mM induced appearance for three new bands with molecular weights 109, 85and 21 KDa with band intensity 5.705, 2.195 and 48.649 %, respectively. Two bands with molecular weight (63 and 25) were disappeared. These treatments induced qualitative and quantitative protein band intensity. These changes observed in appearance new bands, disappearance of some bands and protein bands intensity. Five new bands were appeared with molecular weights 109, 85, 56, 49 and 21 KDa one band disappeared with molecular weights 63 KDa that are as selective markers in these treatments in Nicotiana alata plantlets. Data obtained by 20 Gy and 150& 200 mM NaCl showd that all plantlets were died. The combined effects between dose 25 Gy and NaCl concentrations 50, 100, 150 and 200 mM NaCl were studied. Only one treatment that grow with dose 25 and salt 50 mM.

The dose 25 and 50 mM NaCl induced increasing the number of bands from 8 to 10 bands and appearance at three new bands with molecular weights 109, 61 and 21 KDa with band intensity; 3.44 and

111 Table (21): Comparative analysis of the combined effect of gamma radiation dose 20 & 25Gy and salinity on molecular weight and protein band intensity in Nicotiana alata plantlets using SDS –PAGE.

NaCl concentration (mM) Cont.hap M.W. of Cont.dip. 20 × 50 20×100 25 × 50 Band . M.W M.W M.W M.W M.W

118

114

109 11.083 4.519 2.705 3.44

105 91 14.849

85 4.443 2.195

73 21.765

63 10.14 13.30 61 5.6

56 4.368

49 1.665

44 2.851 4.46 3.382 3.6 41 2.252 4.28 2.677 2.661 3.1

37 6.935 4.04 1.98 5.126 2.3

34 13.36 32.04 7.314 6.0

31 24.41 19.015 24.5

29 6.604 10.80 17.70 15.5

25 22.491 68.99 10.688 10.395 10.7

21 23.612 48.649 5.05

Total .No. 10 8 9 9 10 of band

112

5.05 %; respectively. One band with molecular weights (63 KDa) was disappeared (Table 21). The SDS-polyacrylamide gel system we used to allow separation of the proteins according to their apparent M.W. Changes in the electrophoretic patterns following hardening should therefore reflect variations in the relative abundance of polypeptides with possible splitting of some biochemical modifications on the polypeptides such as methylation, phosphorylation, or conformation changes may escape detection in on system. Detection of such changes was nor desirable because, although protein alterations are probably occurring in the hardening process, they are also part of the normal metabolism of the cell changes in the M.W. instead reflect synthesis or cleavage of proteins and is easier to interpret in terms of appearance of cryaprotective proteins Cloutier (1983). Osmotic adjustment in plants subjected to salt stress can occur by the accumulation of high concentrations of either inorganic ions or low molecular weight organic solutes. Although both of these play a crucial role in higher plants grown under saline conditions, their relative contribution varies among species, among cultivars and even between different compartments within the same plant Ashraf (1994a). The compatible osmolytes generally found in higher plants are low molecular weight sugars, organic acids, polyols, and nitrogen containing compounds such as amino acids, amides, imino acids, ectoine (1,4,5,6-tetrahydro-2- methyl- 4-carboxylpyrimidine), proteins and quaternary ammonium compounds. According to Popp and Smirnoff (1995), of the various organic osmotica, sugars contribute up to 50% of the total osmotic potential in glycophytes subject to saline conditions. The accumulation of soluble carbohydrates in plants has been widely reported as a response to salinity or drought, despite a significant decrease in net CO2 assimilation rate Murakeozy et al. (2003), and examples of changes in carbohydrate concentrations in response to salt stress. Several studies have attempted to relate the magnitude of changes in soluble carbohydrate to salinity tolerance.

113 Ashraf and Tufail (1995) determined the total soluble sugars content in five sunflower accessions differing in salt tolerance. They found that although sugarcontent increased significantly in all five lines with increasing salt in the growth medium, the salt tolerant lines had generally greater soluble sugars than the salt sensitive ones. In contrast, in safflower the pattern of accumulation of soluble sugars differed, even within salt tolerant accessions, since one salt tolerant line accumulated a high content of soluble sugars whereas another line was salt tolerant despite solute accumulation similar to that in salt sensitive accessions Ashraf and Fatima (1995). While comparing salt tolerant wild populations with cultivated populations of Melilotus indica and Eruca sativa, Ashraf (1994b) found that the former had significantly higher soluble sugars in their leaves than the latter salt sensitive populations at varying salt levels of the growth medium. Several salt-induced proteins have been identified in plants species and have been classified into two distinct groups Mansour (2000); salt stress proteins, which accumulate only due to salt stress, and stress associated proteins, which also accumulate in response to heat, cold, drought, waterlogging, and high and low mineral nutrients. Proteins that accumulate in plants grown under saline conditions may provide a storage form of nitrogen that is re-utilized when stress is over (Singh, 1987) and may play a role in osmotic adjustment. Proteins may be synthesized de novo in response to salt stress or may be present constitutively at low concentration and increase when plants are exposed to salt stress (Parida et al., 2002). During characterization of salt-induced proteins in tobacco, a 26 kDa protein named as osmotin was detected (Singh, 1987). In salt stressed Mesembryanthemum crystallinum it was found that osmotin-like protein was increased relative to non-stressed plants (Thomas et al, 1992). In barley, two 26 kDa polypeptides, not immunologically related to osmotin, identified as germin, were increased in response to salt stress (Hurkman et al, 1991). Moreover Lopez et al. (1994) found a 22 kDa protein in response to salt stress in radish. In finger

114 millet (Eleusine coracana), Uma et al. (1995) found 54 kDa and 23– 24 kDa proteins responsible for salt or drought tolerance. While assessing the combined effect of salinity and boron on wheat, Wimmer et al, (2003) reported that the combined stresses induced quantitative and qualitative changes in inter-, but not intracellular protein composition. Most prominent was the induction of a 25 kDa protein and an increase in amount of a 33 kDa protein. They ascribed these changes to structural modifications of the cell wall. In another study, two transgenic rice plants expressing a wheat LEA group 2 protein (PMA80) gene or the wheat LEA group 1 protein (PMA1959) gene were generated (Cheng et al., 2002). Immunoblot analysis exhibited the presence of the LEA group 2 protein (39 kDa) and the LEA group 1 protein (25 kDa) in most of the plant lines. When the second-generation transgenic plants were subjected to dehydration or salt stress, they showed high accumulation of either MA80 or PMA1959, which correlated with increased tolerance of transgenic rice plants to these stresses. While investigating the mechanisms of salt tolerance in a mangrove, Bruguiera sexangula. Yamada et al. (2002) found a specific protein, allene oxide cyclase (AOC) responsible for enhanced salt tolerance. They designated this protein as “mangrin”. Furthermore, expression of mangrin in Saccharomyces cerevisiae and tobacco cell lines also enhanced salt tolerance in these species. A higher content of soluble proteins has been observed in salt tolerant than in salt sensitive cultivars of barley (Hurkman et al. 1989), sunflower Ashraf and Tufail (1995) finger millet (Uma et al. 1995). In wheat, Ashraf and O’Leary (1999) reported that total soluble proteins increased due to salt stress in all cultivars tested but this increase was more marked in a salt sensitive cultivar, Potohar, and low in a salt tolerant line, S24, compared with the other lines. Patterns of polypeptides in wheat cultivars were identical and the differences between cultivars under salt stress were only quantitative. For example, 29 and 48 kDa polypeptides were reduced significantly in salt sensitive cv. Potohar due to salt stress. However, the

115 quantitative changes in polypeptides may be responsible for adjustments in metabolic pathways under saline conditions (Sarhan and Perras, 1987) .In contrast, in lentil, Ashraf and Waheed (1993) reported that leaf soluble proteins decreased due to salt stress in all lines, irrespective of their salt tolerance. Ashraf and Fatima (1995) found that salt tolerant and salt sensitive accessions of safflower did not differ significantly in leaf soluble proteins. Similarly, comparison of salt tolerant wild populations with cultivated populations of Melilotus indica and Eruca sativa. Crowe and Crowe (1984) showed that the salt tolerant populations did not differ from salt sensitive populations in soluble protein content of their leaves at varying salt levels of the growth medium. Although. Pareek et al. (1997) suggested that stress proteins could be used as important molecular markers for improvement of salt tolerance using genetic engineering techniques, in many studies the proteins produced under salt stress are not always associated with salt tolerance. Thus using proteins as salt tolerance indicators depends on the nature of the plant species or cultivar.

116 2- Randomly amplified polymorphic DNA markers (RAPD) Total genomic DNA from ten samples Nicotiana alata were used as templates for RAPD finger printing cultivars.(table, 22). Five random decamer primers were used in this study. Table (23a) shows that the primer OP-C13 gave 9-11 fragments with the ten cultivars, the ten cultivars gave the same fragments with M.W; 990, 820, 740, 680, 550, 440, 300 and 180 Pb. While the diploid only have 1140 pb fragment with absence of 980 pb fragment controlling with other haploid cultivars. Data in (table 23a & Fig 15a) showed that the combined effect of radiation and NaCl were more in total fragments with this primer (11 fragments). The Op-Do7 primer was gave about the same results with the ten cultivars (table 23b & Fig15b), thire 10 fragments were obtained by DNA- RAPD, fragment with 980 pb was absent in the combined cultivars. The OP-L12 primer was gave the lowest total fragments (1-6), this data were clearly that primer OP-L12 was not specific for radiation or salt tolerance genes (table 23c & Fig.15c). Data obtained by using OP-L15 were showed the highly specify for radiation and salt effective genes, it gave about (9-13) fragments with the ten's cultivars DNA. The diploid cultivar was only have not the 930 pb fragment, and the combined effect was the same in the presence of fragments (table 23d & Fig.15d). Table (23e) and Fig. (15 e) gave the DNA RAPD of primer Op-M20 results; it's clearly from these results that total fragments found were 9 or 10. The combined effect samples gave the same results, also the minimum total fragments (7) was obtained by 20 Gy and free salts. Generally, results in table (23) and Fig. (15) showed that the number of fragments amplified by each primer ranged from 1 – 13 with an average of 8.9 fragments per primer. Maximum numbers of bands are (13). Fig. (15c) was produced by the primer OP- L12, whereas minimum number of bands (1) was produced by the primer OP - L15 (Fig. 15d). A representative RAPD profile obtained with primer OP-L15 and OP – M20 (Fig. 15d & e).

117

Table (22). DNA- RAPD of cultivares samples, ploidy and its source.

Sample Ploidy sources No. 1 Diploid Free rad. & salt (as control). 2 Haploid Free rad. & salt (as control). 3 Haploid 15 Gy rad. and salt free. 4 Haploid 20 Gy rad. and salt free. 5 Haploid 25 Gy rad. and salt free. 6 Haploid Free rad. and 150mM NaCl. 7 Haploid Free rad. and 200mM NaCl. 8 Haploid 20 Gy rad. and 50 mM NaCl. 9 Haploid 20 Gy rad. and 100 mM NaCl. 10 Haploid 25 Gy rad. and 50 mM NaCl.

118 Table (23): DNA polymorphism using randomly mplified polymorphic DNA (RAPD) for the ten cultivars with the five primers.

Cultivars Band M.W primer NO. (bp) 1 2 3 4 5 6 7 8 9 10

1 1140 1 0 0 0 0 0 0 0 0 0 2 990 1 1 1 1 1 1 1 1 1 1 3 980 0 1 1 1 1 1 1 1 1 1 4 965 0 1 1 0 0 0 1 1 1 1 OP-C13 5 950 0 1 1 0 0 0 1 1 1 1 6 820 1 1 1 1 1 1 1 1 1 1 7 740 1 1 1 1 1 1 1 1 1 1 8 680 1 1 1 1 1 1 1 1 1 1 9 550 1 1 1 1 1 1 1 1 1 1 10 440 1 1 1 1 1 1 1 1 1 1 11 300 1 1 1 1 1 1 1 1 1 1 12 180 1 1 1 1 1 1 1 1 1 1 Total 9 11 11 9 9 9 11 11 11 11

(b)

cultivars Band M.W Primer NO. (bp) 1 2 3 4 5 6 7 8 9 10

1 980 0 0 1 1 0 1 0 0 0 0 2 940 1 1 1 1 1 1 1 1 1 1 3 915 1 1 1 1 1 1 1 1 1 1 4 820 1 1 0 1 1 1 1 1 1 1 OP-D07 5 680 1 1 1 1 1 1 1 1 1 1 6 540 1 1 1 1 1 1 1 1 1 1 7 490 1 1 1 1 1 1 1 1 1 1 8 450 1 1 1 1 1 1 1 1 1 1 9 360 1 1 1 1 1 1 1 1 1 1 10 350 1 1 1 1 1 1 1 1 1 1 11 230 1 1 1 1 1 1 1 1 1 1 Total 10 10 10 11 10 11 10 10 10 10

119 Table (23).Cont. (c)

Cultivars M. Band W No. (bp) 1 2 3 4 5 6 7 8 9 10 primer 1 1120 0 1 0 0 0 1 0 0 0 0 2 680 0 1 1 1 1 1 1 0 0 0 OP-L12 3 570 0 1 1 1 1 1 1 0 0 1 4 460 1 1 1 1 1 1 1 0 0 1 5 440 1 1 1 1 1 1 1 1 1 1 6 300 1 1 1 1 1 1 1 1 0 1 Total 3 6 5 5 5 6 5 2 1 4

(d) Cultivars

Band M.W

primer No. (bp)

1 2 3 4 5 6 7 8 9 10 1 1290 1 1 1 1 1 1 1 1 1 1 2 960 1 1 1 0 1 0 1 1 1 1 3 945 1 1 1 1 1 1 1 1 1 1 4 930 0 1 1 1 1 1 1 1 1 1 5 885 1 1 0 0 1 0 1 1 1 1 6 830 1 1 1 1 1 1 1 1 1 1 OP-L15 7 700 1 1 1 0 1 1 1 1 1 1 8 545 1 1 1 1 1 1 1 1 1 1 9 490 1 1 1 1 1 1 1 1 1 1 10 425 1 1 1 1 1 1 1 1 1 1 11 380 0 0 1 1 0 1 1 1 1 1 12 340 1 1 1 1 1 1 1 1 1 1 13 230 1 1 1 0 1 1 1 1 1 1

Total 11 12 12 9 12 11 13 13 13 13

120 Table (23).Cont.

(e)

Cultivars primer Band M.W No. (bp) 1 2 3 4 5 6 7 8 9 10

1 1360 1 1 1 0 0 1 1 1 1 1 2 915 1 1 1 1 1 1 1 1 1 1 3 890 1 1 1 0 1 1 1 1 1 0 4 800 0 0 1 0 0 1 0 1 1 1 5 715 1 1 1 1 1 1 1 1 1 1 OP-M20 6 525 0 0 0 1 1 0 1 1 1 1 7 460 1 1 1 1 1 1 1 1 1 1 8 420 1 1 1 0 1 0 1 1 1 1 9 400 1 1 1 1 1 1 1 1 1 1 10 385 1 1 1 1 1 1 1 1 1 1 11 340 1 1 1 1 1 1 1 1 1 1 12 290 0 0 0 0 1 0 0 0 0 0

Total 9 9 10 7 10 9 10 11 11 10

121

OP-C13 OP-D07

(a) (b)

OP-L12 OP-L15

(c) (d) OP-M20

(e)

Fig. (15): RAPD patterns of Nicotiana alata treatments generated by primers; a) OP -C13, b) OP -DO7, c) P -L12, OP -L15 and e) OP -M20.

122 The primer OP- L12 produced maximum number of bands (13) in the salinity and the combined effect of salinity and gamma radiation. Also in radiation treatments the same primer produced the maximum number of bands (12). The minimum number of bands was observed with primer OP- I15 (3, 3 and1) in the irradiated, salt stress and combined effect of gamma radiation and salt stress plants, respectively. Table (24) gave the results obtained by all the five primers as a groubs effect; Radiation, Salinity and the combined effects; to producing polymorphic bands in each groub and its percentage, Data in this table suggested that all the primers were polymorphic. A total of 270 bands were amplified of which 226 (83.7 %) polymorphic across the radiation treatments. The level of polymorphism among the salinity stress was 85.6 %. Whereas, the polymorphism among the combined effects of gamma radiation and salinity was 85.5% (Table 24). Results obtained by the five primers determined that all the primers except OP- L12 were gave specific markers , the total of 18 specific markers (table 25) were generated; radiation, and salinity and the combined effect between gamma irradiation and salinity were generated ; 5,6 and 3 specific markers.

Among the 10 treatments were clearly differentiated by producing one or two specific bands with all the five primers used.

123 Primer Treatments No. Radiation (3,4&5) Salinity (6&7) salinity× Radiation(8,9&10) Total No Total No Total No No. polymorphic No. Polymorphic No. polymorp band band band band band hic band OP-C13 60 49(81.7%) 48 40(83.3%) 60 53(88.3% ) OP-D07 55 51(92.77%) 44 41(93.2%) 55 50(90.9% ) OP-I15 30 24(80%) 24 20(83.3%) 30 16(53.3% ) OP-L12 65 57(87.77%) 52 47(90.4%) 65 62(95.4% ) OP- 60 45(75%) 48 37(77.1%) 60 50(83.3% M20 ) Total 270 226(83.7%) 216 185(85.6%) 270 231(85.51 %)

Table (24): RAPD patterns of Nicotiana alata treatments generated by the five primers

ﺗﻢ اﻟﺘﺼﻠﯿﺢ ﻓﻰ ﺟﺪاول ﺑﺎﻟﻌﺮض

124 Table (25):Rad. and salt combined effect for each primer and its specific markers in the ten cultivars.

Primer No. band haploid Diploid Rad. Salt. Combined OP-C13 1140 1 0 0 0 0 OP-D07 980 0 0 1 0 0

OP-L15 1120 0 1 0 1 0 680 0 1 1 1 0 OP-L12 930 0 1 1 1 1

800 0 0 1 1 1 OP- M20 525 0 0 1 1 1 290 0 0 0 1 0

Total specific 1 3 5 6 3 marker

Genetic relationships in treatments Common bands were scored as present ambiguous, or absent and the data used to calculate values of genetic distance between all the treatments studied. The results are presented in table (26). The genetic distance scale runs from 0 (identical) to100 (different for all criteria studied).The similarity index as shown in this table (26) revealed that the maximum similarity of diploid with irradiated haploid with dose15Gy (similarity indices 1.0) while distantly related treatments were (20Gy×50) m mol with (20Gy×100) (similarity indices 0.0). The relationships within radiation treatments were 54%, however these relationships within salinity treatments were 49%, while the relationships with the combined treatments between salinity and gamma radiation were 17-18%.

125 Table (26): Similarity index (Pairwise comparison) among the ten cultivars based on RAPD analysis. 20Gy 20Gy 25Gy Control Control 15 150 200 20Gy 25Gy 50 × × 100 × 50 Treatments diploid haploid Gy mM mM mM mM mM Control diploid Control 0.46 haploid 15 Gy 0.72 0.30 20Gy 1.0 0.79 0.66 25Gy 0.47 0.31 0.56 0.54 150 mM 0.73 0.43 0.31 0.41 0.57 200 mM 0.51 0.11 0.24 0.59 0.24 0.49 20Gy × 50 0.53 0.37 0.37 0.87 0.50 0.63 0.18 mM 20Gy × 100 0.60 0.43 0.43 0.95 0.57 0.70 0.24 0.0 mM 25Gy × 50 0.59 0.30 0.30 0.66 0.43 0.56 0.11 0.12 0.17 mM

Figure (16): A dendrogram showing the genetic distance among the ten treatments using RAPD data.

0.0 0.2 0.4 0.6 0.8 1.0

126 Fig. (16) gave a dendrogram for the genetic relationships between the ten cultivars, this figure suggest that all the treatments of haploid Nicotiana alata have been separated into three main cluster revealed that one cluster consisted of combined treatments (salinity & gamma radiation) and salinity treatments, second one that of gamma radiation treatments, with diploid and haploid plants and the third one that of irradiated plants with dose (25Gy) and the haploid plants grow on (50 ml mol) NaCl. The treatments and the similarity between them in the first cluster was (0.11), in the second cluster was (0.56) and in third cluster was (0.57) Fig. (16). The arbitrary nucleotide sequences, RAPD finger printing is frequently used technique for investigating genetic polymorphisms (Versalovic et al. 1994). Teaumroong and Boonkerd (1998). RAPD markers have been used for numerous applications in plant molecular genetic research despite having disadvantages of poor reproducibility and not generally being associated with gene regions (Welsh and McClelland. 1990 and Williams et al, 1990). RAPD, being a multi locus marker (Karp et al. 1997) with the simplest and fastest detection technology, have been successfully employed for determination of interspecies genetic diversity. Characterization and quantification of genetic diversity has long been a major goal in breeding. In plant breeding programs, information on the genetic diversity within and among closely related crop species is essential for a rational use of genetic resources. The result showed that RAPD assay discriminate those flue-cure tobacco cultivars with similar genotypes. It seems that RAPD is an effective tool for flue-cured tobacco germplasm management, cultivar protection, and cultivar improvement. The fragment polymorphism was higher than the reported in case of egg plant (10.28 bands per primer ), which also belongs to the family solanaeceae (Singh et al. 2005).The number of fragments amplified in N. tabacum was significantly higher than that reported earlier (Del piano et al. 2000). The high degree of genetic polymorphism among the 7 species of Nicotiana was also reported using AFLP (Ren and Timko, 2002). In contrast, genetic diversity analysis by RAPD markers revealed 79% polymorphism in the species of L. peruvianum and very low (9%) in the species L.

127 peruvianum of the genus Lycopersicon (Kochieva et al. 2002). Species –specific RAPD patterns have been developed and used to confirm hybridity in potato (Takemori and Kadotini 1994) and intergeneric hybrids of sacharum and Erainthus (Nair et al. 2002). Good speed (1954) had postulated that the present day ass emblage of species was derived froma pregeneric genetic reservoir with three major components that had been designated pre- Nicotiana, pre-Cestrum and pre-petunia. The Cestroid complex was thought to be ancestral to the Rustica whereas petunioides complex ancestral to the subgenus petunioides. Grouping of these species was clearly in accordance with the earlier classification based on traditional analysis of cytological and morphological characteristics (Goodspeed, 1954). In conclusion,the present study would be useful for establishing molecular phyloeny in the species of Nicotiana. RAPD assay was also found effective in analyzing polymorphism at the subspecies level. The species- specific markers identified would be utilized in introgression breeding programmes. Randomly amplified polymorphic DNA markers (RAPD): In 1990, Williams et al. developed a new PCR-based genetic assay namely randomly amplified polymorphic DNA (RAPD). The standard RAPD technology utilizes short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. mplification products are generally separated on agarose gels and stained with ethidium bromide (Williams et al. 1990). On an average, each primer directs amplification of several discrete loci in the genome, making the assay useful for efficient screening of nucleotide sequence polymorphism between individuals. Since RAPD amplification is directed with a single, arbitrary and short oligonucleotide primer, DNA virtually from all sources is amenable to amplification. However, due to the stoichastic nature of DNA amplification with random sequence primers, it is important to optimize and maintain consistent reaction

128 conditions for reproducible DNA amplification. RAPD markers are dominant markers and have found a wide range of applications in gene mapping, population genetics, molecular evolutionary genetics and plant and animal breeding. This is mainly due to the speed, cost and efficiency of the RAPD technique to generate large numbers of markers in a short period compared with previous methods. Therefore, RAPD technique can be performed in a moderate laboratory for most of its applications. Because the chance of comigrating bands being homologous becomes less as populations diverge, it was suggested that RAPD analysis gives more accurate estimates between closely related populations and less accurate estimates for distantly related populations (Williams et al. 1993). Despite problems such as poor reproducibility faint or fuzzy products, and difficulty in scoring bands, the RAPD method will probably be important as long as other DNA-based techniques remain unavailable in terms of cost, time and labour.

Applications of molecular in tobacco genome analysis and breeding Molecular markers have been looked upon as tools for a large number of applications ranging from localization of a gene to improvement of plant varieties by marker-assisted selection. They have also become extremely popular markers for phylogenetic analysis adding new dimensions to the evolutionary theories. If we look at the history of the development of these markers, it is evident that they have been improved over the last two decades to provide easy, fast and automated assistance to scientists and breeders. Genome analysis based on molecular markers has generated a vast amount of information and a number of databases are being generated to preserve and popularize it. Evolutionary genetics The advances in DNA techniques have had a great impact in addressing problems in many aspects of biology.Comparative biochemical studies and examinations oforganellar (plastid and mitochondrial) genome organizationsupport the viewpoint that

129 Nicotiana sylvestris is the maternal parent and Nicotiana tomentosiformis the paternal parent (Sperisen et al. 1991). Analysis of molecular features, such as repetitive DNA sequences and the structure of various nuclear gene family members, has also been employed to trace the molecular evolution of tobacco and study the genetic diversity in genus Nicotiana (Kuhrová et al., 1991; Komarnitsky et al., 1998 and Lim et al. 2000a & b). In most cases, these studies also concluded that N. tomentosiformis was the progenitor parental donor along with N. sylvestris. Based on investigations of the structure and organization of mitochondrial and chloroplast genomes, (Olmstead and Palmer, 1991). Demonstrated that a species similar to N. sylvestris donated the maternal genome of tobacco. Lim et al. (2000a) suggested that the majority of the genes present in N. tabacum were contributed by N. sylvestris, with the remainder being contributed by N. tomentosiformis. Pairwise comparisons of the AFLP profiles of wild and cultivated Nicotiana species show that polymorphic bands present in N. tabacum can be found in at least one of three proposed wild progenitor species (i.e., N. sylvestris, N. tomentosiformis, and Nicotiana otophora), and this observation provides additional support for these species contributing to the origin of N. tabacum (Ren and Timko, 2002).

Population genetics and genotyping of cultivars Application of DNA-based approaches to population genetic studies has been limited, probably due to the need for large samples of individuals from each population to provide an accurate estimate of allele and genotype frequencies. The relatively high cost, therequirement for sophisticated equipment and well-trained personnel, and low speed are other limiting factors in population genetic studies. The RAPD technique has received a great deal of attention from population geneticists because of its simplicity and rapidity in revealing DNA-level genetic variation, and therefore has been praised as the DNA equivalent of allozyme electrophoresis (Hedrick, 1992 and Skibinski, 1994). Numerous types of tobacco are grown

130 commercially and are defined to a large extent by region and (or) area of production, method of curing, and intended use in manufacturing, as well as by some distinct morphological characters and chemical differences (Wernsman, & Rufty, 1987). At the present time, only limited information is available on the relationship between morphological variability and genetic diversity in cultured tobacco. Attempts have been made to examine the degree of relatedness between tobacco cultivars and types at the molecular genetic level. The repetitive and arbitrary DNA markers are markers of choice in genotyping of cultivars. Zhang et al. (2005) employed the RAPD analysis to assess the polymorphism, similarities and relationships among N. tabacum L. cultivars. One hundred and forty-nine bands were detected, of which 94 were polymorphic (63.1%). A primer distinguishing all of the tested cultivars was found. The result showed that RAPD assay could discriminate those flue-cure tobacco cultivars with similar genotypes. Genetic diversity and genotyping in Indian FCV and burley tobacco cultivars was also investigated by Saraka and Rao using RAPD technique (Saraka and Rao, 2008). In these studies, the markers found specific to the varieties can be used in correct identification of the carrier genotypes in trade and commerce.

Mapping and tagging of genes Diversity analysis of germplasm Germplasm analysis to study genetic diversity is another important area in which a lot of efforts have been put in. Characterization and quantification of genetic diversity has long been a major goal in breeding. In plant breeding programs, information on genetic diversity is essential for a rational use of genetic resources. It is particularly useful in characterizing individual accessions and cultivars, in detecting duplications of genetic materials in germplasm collections, and as a general guide in selecting parents for hybridization in breeding programs and in developing informative mapping populations for genome mapping. Studies revealed that cultured tobacco groups were divided primarily based upon geographic origin and manufacturing quality

131 traits (Ren and Timko, (2002) and Zhang et al., 2006 & 2008). The amount of genetic polymorphism present among cultivated tobacco lines (N. tabacum) was limited (Ren and Timko, 2002; Zhang et al., 2006 & 2008 and Siva-Raju et al., 2008), as evidenced by the high degree of similarity. A greater amount of genetic polymorphism exists among wild species of Nicotiana than among cultivated forms (Ren and Timko, 2002). The high degree of genetic polymorphism among species that was observed by (Ren and Timko, 2002) using AFLP analysis is consistent with the observations of Bogani et al. (1997) following their analysis of interspecific variation using RAPD analysis. These studies indicated that the present day commonly grown tobacco germplasm has narrow genetic diversity among the cultivars, necessitating a sustained effort to preserve tobacco germplasm resources. In another study,amplified fragment length polymorphism (AFLP) analysis was used to determine genetic variation in 54 varieties of cultivated tobacco (N. tabacum and N. rustica) and three accessions of exotic germplasm.

Perspective of molecular markers’ aplicationin tobacco genetics and breeding Narrow genetic diversity has been revealed among the commonly grown tobacco germplasm (Ren and Timko, 2002 and Zhang et al., 2006 & 2008), and it is possible that a large proportion of valuable tobacco germplasm may already have been lost through the popularity of certain cultivars in commercial planting and the continuous artificial selection (Zhang et al., 2006). Since greater genetic variation exists among wild species of Nicotiana than among cultivated forms (Ren and Timko, 2002), to avoid further degradation of germplasm resources, crosses should be made with genetically distant varieties or wild species. More information of genetic variation within commercially cultivated tobacco between cultivated forms of N. tabacum and its wild relatives should be revealed by molecular markers Species-specific markers identified in studies are useful in identification of the true hybrids and monitoring introgression of useful genes from the wild relatives.

132

SUMMARY The present investigation was under taken on one speciess of tobacum plants (Nicotiana alata); it was carried out in the laboratories of Botany Dept. (genetics), Fac. Of Agri., Al-Azhar Unive., and Natural Product Dept., National Center for Radiation Research and Technology, Atomic Energy Authority, Cairo, to study the haploid plants produced from anther culture of Nicotiana alata, diploidization for haploids by colchicine and the effect of gamma radiation doses, salinity and the combined effects between them on tissue culture were also studied.The molecular genetic markers in Nicotiana alata haploid plants were detected too, Results obtained were; Firest; Haploid plants induction from anther cultures: 1-The best medium used in callus induction and morphology was medium. 2- There media were used for micropropagation media Nicotiana alata, MS medium supplemented 0.2 IAA+0.5 KIN mg/l were found to be the best. The other hormones concentrations induced weak or absent growth and formed callus. 3 -The best medium for regenerated the anther callus was (Ms +1.0BAP +0.5NAA). 4- Haploid plantlets were examined for haploidy by;

a) Staining the root tips squshes for chromosome number and found that each cell contains 9 chromosomes. b) Morphological compairing between the haploid and diploid plantlets were gave a different shapeis for leaves and flawers sises. c) Pollen grain activities for haploid plants were examined also by staining and compairinig with diploid; haploid pollen was less in activity. d) Diplodization for haploid by 0.7% cholichine pure diploid plants like haploid in shapes.

133

Second; The effect of gamma radiation and Salinity on haploid plants; 1- Gamma radiation doses induced decreasing in callus growth rate in Nicotiana alata plants with increasing gamma radiation dose 20 Gy was the most effective dose on while 25 Gy was the death dose. 2- The effect of gamma radiation doses induced decreasing in the anther and bud surivival percentage with increasing radiation dose in Nicotiana alata. 3- The saline concentrations induced decreasing in bud survival and shoot length with increasing NaCl /L concentrations, 250 mM NaCl was the death concentration. 4- The combined effects between gamma radiation doses and NaCl concentrations on the buds survival percentage were decreased with increasing doses and concentrations. Untile leades to 100 mM with 25 Gy which gave zero. 5- The effect of gamma radiation, salinity and the combined effects between them on total protein and proline contents were found as follows; a) The use of different gamma radiation doses induced changing in total protein in Nicotiana alata haploid plants.some doses (2.5 and 5 Gy) induced increasing in total protein, while its decreased in the other doses (20 and 25Gy). b) The proline contents in Nicotiana alata plants were increased with increasing of gamma radiation doses, the highest (1.50 mg prolein / 100 g) fresh weight was obtained by 25Gy. c) The salt levels were increased the total protein by increasing salt dose, Nicotiana alata plants induced increaseing in total protein untile 2.24 mg /100 g fresh weight on 200 mM Na Cl. d) The proline content was increased with increasing salt concentration from 50 to 200 mM Na Cl (1.55 to 1.88 mg proline 100 g fresh weigth).

134

e) The combined effects between gamma radiation doses, and NaCl concentrations in Nicotiana alata induced increasing in proline content, the 10 Gy + 50 mM Na Cl gave the lowest proline contents, while 15 Gy + 200 mM Na Cl gave the highest proline contents in plants (1.08 mg /100 g fresh weight). f) The combined effects of gamma radiation and NaCl concentrations on total protein were fluctuate in Nicotiana alata plants, some doses induced increasing (10 Gy + 150 mM NaCl concentration) but the other treatments due to decreesing in total protein contents. Third; Moluclar markers by using SDS-PAGE. 1- Gamma radiation doses (0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy) were induced qualitative and quantitative differences represented in appearance, disappearance or changes in some bands intensity.compairing with diploid parents: 2- There was one band disappeared with molecular weight (44 KDa), in irradiated Nicotiana alata considerable as a negative selective markers. 3- Salinity effect induced qualitative and quantitative changes in protein bands of haploid plants. Ther was one band with molecular weight (63 KDa) disappeared. Which can be used as a detectable marker in plantlets grew under salinity stress. 4- The combined effects between gamma radiation doses and salinity induced quantitative and qualitative changes as: a) The combined effects of dose 10 Gy and 50, 100,150 and 200 ml mol NaCl induced two bands disappeared with molecular weight (44 KDa). b) The combined effects of dose 15 Gy with NaCl concentrations (50, 100,150 and 200 ml mol) induced disappeared for one band with molecular weight (63 KDa).

135

c) The combined effects between dose 20 Gy and 50, and 100 mM NaCl induced two new bands which appeared with molecular weight (85&109 KDa) and one band disappeared with molecular weights (63 KDa). d) The combined effects of dose (25Gy and 50 mM NaCl) there were three new bands appeared with molecular weights (21, 61& 109 KDa respectively) and one band disappeared with molecular weight (63 KDa,), these foure bands were used as a selective molecular marker for haploid plants. Fourth; Molecular finger print determination by using RAPD- PCR; Five primers were used for determination genetical finger prents by the RAPD-PCR technique, results obtained were; 1- By using the primer OP-C13 and OP-L12, the haploid plants were gave disappearing for the band (1140bp) and appearing for a new band (930 bp), respectively as a molecular indicator for the combined effect between radiation and salinity. 2- The primer OP-DO7 gave a new band (980 bp); this band can used as a indicator for radiation. 3- The OP-L15 primer gave also a new band (1120 bp) with the salinity treatments. 4- The same results for the primer OP-M20 which gave two bands (525 & 800 bp) as a molecular indicator for each radiation dose, salinity concentration and it’s the combined effects.

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173 ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﯿﻢ اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ أﺟﺮى اﻟﺒﺤﺚ ﻋﻠﻰ أﺣﺪ أﻧﻮاع ﻧﺒﺎﺗﺎت اﻟﺪﺧﺎن وھﻰ ﻧﺒﺎﺗﺎت دﺧﺎن اﻟﺰھﻮروذﻟﻚ ﺑﻤﻌﺎﻣﻞ ﻗﺴﻢ اﻟﻨﺒﺎت اﻟﺰراﻋﻰ (اﻟﻮراﺛﺔ) - ﻛﻠﯿﺔ اﻟﺰراﻋﺔ – ﺟﺎﻣﻌﺔ اﻷزھﺮ- ﺑﺎﻟﻘﺎھﺮة وﻗﺴﻢ ﺑﺤﻮث اﻟﻤﻨﺘﺠﺎت اﻟﻄﺒﯿﻌﯿﺔ – اﻟﻤﺮﻛﺰ اﻟﻘﻮﻣﻰ ﻟﺒﺤﻮث وﺗﻜﻨﻮﻟﻮﺟﯿﺎ اﻹﺷﻌﺎع – ھﯿﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﯾﺔ – ﺑﺎﻟﻘﺎھﺮة وذﻟﻚ ﺑﮭﺪف اﻧﺘﺎج اﻟﺤﺎﻟﺔ اﻷﺣﺎدﯾﺔ ودراﺳﺔ أﻟﺘﺄﺛﯿﺮات اﻟﺤﺎدﺛﺔ ﻟﻠﻨﺒﺎﺗﺎت أﻷﺣﺎدﯾﺔ ﺑﻌﺪ ﻣﻀﺎﻋﻔﺘﮭﺎ ﻛﺮوﻣﻮزوﻣﯿﺎ ﻋﻠﻰ اﻟﻤﺴﺘﻮى اﻟﺠﺰﯾﺌﻰ ﺑﺎﺳﺘﺨﺪام اﻟﺘﻘﻨﯿﺎت اﻟﺤﺪﯾﺜﺔ، ﻣﻊ دراﺳﺔ أﻟﺘﺄﺛﯿﺮات اﻟﺤﺎدﺛﺔ ﻟﻤﻌﺎﻣﻠﺔ اﻷﺷﻌﺎع اﻟﺠﺎﻣﻰ وأﻟﺘﺄﺛﯿﺮ اﻟﻤﻠﺤﻰ ﻟﻜﻠﻮرﯾﺪ اﻟﺼﻮدﯾﻮم ﻋﻠﻰ اﻟﻤﺴﺘﻮى اﻟﺠﺰﯾﺌﻰ ﻟﻠﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ ﻣﻊ ﻣﻘﺎرﻧﺘﮭﺎ ﺑﺎﻷﺑﺎء اﻟﺜﻨﺎﺋﯿﺔ. وﻗﺪ ﺗﻢ اﻟﺘﺤﺼﻞ ﻋﻠﻲ اﻟﻨﺘﺎﺋﺞ اﻷﺗﯿﺔ:- أوﻻ:اﺳﺘﺤﺪاث اﻟﺒﺎدرات اﻷﺣﺎدﯾﺔ ﻣﻦ ﻣﺰارع اﻟﻤﺘﻚ. 1- ﺳﺘﺤﺪاث اﻟﻜﺎﻟﻮس اﻷﺣﺎدى ﻓﻰ ﻣﺰارع اﻟﻤﺘﻚ وﻗﺪ أﺳﺘﺨﺪم ﻓﯿﮭﺎ اﻟﻌﺪﯾﺪ ﻣﻦ اﻻﺿﺎﻓﺎت ﻋﻠﻰ ﺑﯿﺌﺔ MS وﻛﺎن أﻓﻀﻞ اﺿﺎﻓﺔ ھﻰ (0.4mgNAA+ 0.5mg KIN) . 2- ﺗﻢ اﺳﺘﺨﺪم ﺛﻼث ﺑﯿﺌﺎت ﻷﻧﺘﺎج ﺑﺎدرات أﺣﺎدﯾﺔ ﻓﻰ ﻣﺰارع اﻟﻤﺘﻚ وأﻋﻄﺖ اﻟﺒﯿﺌﺔ MS + (0.2mg IAA+ 0.5mg KIN) أﻓﻀﻞ اﻟﻨﺘﺎﺋﺞ اﻟﻤﺘﺤﺼﻞ ﻋﻠﯿﮭﺎ. 3- ﻻﻧﺘﺎج اﻟﺘﻜﺸﻒ واﻧﺘﺎج ﻧﺒﯿﺘﺎت ﻛﺎﻣﻠﺔ ﻣﻦ اﻟﻜﺎﻟﺲ أﻷﺣﺎدى وﻛﺎن أﻓﻀﻞ ﺑﯿﺌﺔ MS ﻣﻀﺎف إﻟﯿﮭﺎ (1.0mg BAP + 0.5 NAA) 4- ﺗﻢ اﻟﺘﺄﻛﺪ ﻣﻦ اﻟﺤﺎﻟﺔ اﻷﺣﺎدﯾﺔ ﻓﻰ اﻟﺒﺎدرات و اﻟﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ اﻟﻨﺎﺗﺠﺔ ﺑﻌﺪة ﻃﺮق :- أ- ﻋﻤﻞ ﻓﺤﺺ ﺳﯿﺘﻮﻟﻮﺟﻰ ﻓﻰ ﻗﻤﻢ اﻟﺠﺬور ووﺟﺪ أﺣﺘﻮاء أﻧﻮﯾﺔ اﻟﺨﻼﯾﺎ ﻋﻠﻰ 9 ﻛﺮوﻣﻮزوﻣﺎت وذﻟﻚ ﺑﺎﻟﺼﺒﻎ ﺑﺼﺒﻐﺔ اﻷﺳﯿﺘﻮﻛﺎرﻣﻦ. ب- ﺗﻢ ﻣﻘﺎرﻧﺔ اﻟﻨﺒﺎﺗﺎت ﻣﻮرﻓﻮﻟﻮﺟﯿﺎ ﻣﻊ اﻟﻨﺒﺎﺗﺎت اﻷﺑ ﻮﯾﺔ اﻟﺜﻨﺎﺋﯿﺔ ووﺟﺪ اﺧﺘﻼف ﻓﻰ اﻟﺸﻜﻞ اﻟﺨﺎرﺟﻰ وﺣﺠﻢ اﻷوراق واﻷزھﺎر. ج - ﺑﻔﺤﺺ ﺣﺒﻮب اﻟﻠﻘﺎح ﻟﻠﻨﺒﺎﺗﺎت اﻟﻤﺰھﺮة وﺟﺪ أﻧﮭﺎ ﻓﺎﻗﺪة اﻟﺤﯿﻮﯾﺔ وذات ﺣﺠﻢ أﺻﻐﺮ ﻣﻦ ﺣﺒﻮب ﻟﻘﺎح اﻷﺑﺎء اﻟﻤﻜﺘﻤﻠﺔ اﻟﺤﯿﻮﯾﺔ. د- ﺗﻢ ﻋﻤﻞ ﻣﻀﺎﻋﻔﺔ ﻟﻠﺨﻼﯾﺎ واﻧﺘﺎج اﻟﻨﺒﺎﺗﺎت اﻟﺜﻨﺎﺋﯿﺔ اﻟﺬاﺗﯿﺔ ﺑﺎﺳﺘﺨﺪام ﺗﺮﻛﯿﺰ %0.7 ﻛﻮﻟﺸﯿﺴﯿﻦ ﺑﻮاﺳﻄﺔ اﻻﺿﺎﻓﺔ ﻓﻰ ﺑﯿﺌﺔ اﻟﻨﻤﻮ ﻟﻠﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ ﺛﻢ اﻟﻔﺤﺺ اﻟﺴﯿﺘﻮﻟﻮﺟﻰ ﻟﮭﺎ ووﺟﺪأﻧﮭﺎ ﻣﺸﺎﺑﮭﮫ ﻟﻸﺣﺎدى. ﺛﺎﻧﯿﺎ: ﺗﺄﺛﯿﺮ اﺷﻌﺎع واﻟﻤﻠﻮﺣﺔ ﻋﻠﻰ اﻟﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ: 1- ﺗﺄﺛﯿﺮ ﺟﺮﻋﺎت أﺷﻌﺔ ﺟﺎﻣﺎ ﻋﻠﻰ ﺷﻜﻞ وﻧﻤﻮ اﻟﻜﺎﻟﻮس أﺣﺪﺛﺖ ﺟﺮﻋﺎت أﺷﻌﺔ ﺟﺎﻣﺎ ﻧﻘﺼﺎ ﻓﻰ ﻧﻤﻮ اﻟﻜﺎﻟﻮﺳﺎت ﺑﺰﯾﺎدة اﻟﺠﺮﻋﺔ اﻹ ﺷﻌﺎﻋﯿﺔ وﻛﺎﻧﺖ أﻛﺜﺮ اﻟﺠﺮﻋﺎت ﺗﺄﺛﯿﺮا 20ﺟﺮاى ﻛﻤﺎ ﻛﺎﻧﺖ اﻟﺠﺮﻋﺔ اﻟﻤﻤﯿﺘﺔ ھﻰ 25 ﺟﺮاى. - 2- دراﺳﺔ ﺗﺄﺛﯿﺮ أﺷﻌﺔ ﺟﺎﻣﺎ ﻋﻠﻰ ﺣﯿﻮﯾﺔ اﻟﺒﺮاﻋﻢ ﻟﻠﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ و اﻟﻤﺘﻚ أﺣﺪﺛﺖ اﻟﺠﺮﻋﺎت اﻹﺷﻌﺎﻋﯿﺔ إﻧﺨﻔﺎض ﻓﻰ ﻧﺴﺒﺔ اﻟﺤﯿﻮﯾﺔ ﻟﻠﺒﺮاﻋﻢ ﺑﺰﯾﺎدة اﻟﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ ﻣﻊ

1 اﻧﺨﻔﺎض ﻣﻠﺤﻮظ ﻓﻰ ﻧﺴﺒﺔ اﻟﺤﺼﻮل ﻋﻠﻰ ﻧﺒﺎﺗﺎت أﺣﺎدﯾﺔ ﻣﻦ اﻟﻤﺘﻚ ﺑﺰﯾﺎدة اﻟﺠﺮﻋﺔ اﻷﺷﻌﺎﻋﯿﺔ. 3- وﺟﺪ أن ﺣﯿﻮﯾﺔ اﻟﺒﺮاﻋﻢ وﻃﻮل اﻟﺴﺎق ﻓﻰ اﻟﺒﺎدرات ﺗﺘﻨﺎﻗﺺ ﺑﺰﯾﺎدة اﻟﺘﺮﻛﯿﺰ اﻟﻤﻠﺤﻰ ﺣﺘﻰ ﺗﻮﻗﻒ اﻟﻨﻤﻮ ﻋﻨﺪ ﺗﺮﻛﯿﺰ 250 ﻣﻞ ﻣﻮل ﻛﻠﻮرﯾﺪ ﺻﻮدﯾﻮم. 4- أﻋﻄﻰ اﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﻟﻠﺠﺮﻋﺎت اﻟﻤﺨﺘﻠﻔﺔ ﻣﻦ أﺷﻌﺔ ﺟﺎﻣﺎ وﺗﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ اﻟﻤﺨﺘﻠﻔﺔ ﻋﻼﻗﺔ ﻋﻜﺴﯿﺔ ﺣﯿﺚ ﺗﺄﺛﺮت ﺣﯿﻮﯾﺔ اﻟﺒﺮاﻋﻢ وﺗﻨﺎﻗﺼﺖ ﺑﺰﯾﺎدة اﻟﺠﺮﻋﺎت اﻻﺷﻌﺎﻋﯿﺔ وﺗﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ ﺣﺘﻰ وﺻﻠﺖ اﻟﻰ اﻟﺼﻔﺮ ﻓﻰ ﺗﺮﻛﯿﺰ 100ﻣﻞ ﻣﻮل ﻣﻊ ﻣﻌﺎﻣﻠﺔ اﺷﻌﺎع 25 ﺟﺮاى. 5- ﺑﺪراﺳﺔ ﺗﺄﺛﯿﺮﻛﻼ ﻣﻦ اﻻﺷﻌﺎع اﻟﺠﺎﻣﻰ واﻟﻤﻠﻮﺣﺔ واﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﺑﯿﻨﮭﻤﺎ ﻋﻠﻰ ﻣﺴﺘﻮى ﻛﻼ ﻣﻦ اﻟﺒﺮوﺗﯿﻦ اﻟﻜﻠﻰ واﻟﻤﺤﺘﻮى ﻣﻦ ﺣﻤﺾ اﻟﺒﺮوﻟﯿﻦ وﺟﺪ ﻣﺎ ﯾﻠﻰ:- أ- أﻋﻄﻰ ﻣﺤﺘﻮى اﻟﺨﻼﯾﺎ ﻣﻦ اﻟﺒﺮوﺗﯿﻦ اﻟﻜﻠﻰ ﻣﻊ اﺳﺘﺨﺪام اﻟﺠﺮﻋﺎت اﻻﺷﻌﺎﻋﯿﺔ ﺣﺪوث ﺗﻐﯿﺮات ﻓﻰ ﻣﺤﺘﻮى اﻟﺒﺮوﺗﯿﻦ اﻟﻜﻠﻰ ﻓﻰ اﻟﻨﺒﺎت اﻷﺣﺎدﯾﺔ ﺣﯿﺚ ﺗﺰاﯾﺪ اﻟﻤﺤﺘﻮى اﻟﺒﺮوﺗﯿﻨﻰ ﻓﻰ اﻟﻤﻌﺎﻣﻠﺔ 5 ,2.5 ﺟﺮاى ﺛﻢ ﺗﻨﺎﻗﺺ ﺑﺎﻟﻤﻌﺎﻣﻠﺔ ب25 ,20 ﺟﺮاى. ب- ﻟﺘﺤﺪﯾﺪ ﻣﺤﺘﻮى ﺣﻤﺾ اﻟﺒﺮوﻟﯿﻦ ﻓﻰ ﺧﻼﯾﺎ اﻟﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ أﺳﺘﺨﺪﻣ ﺖ اﻟﺠﺮﻋﺎت اﻹﺷﻌﺎﻋﯿﺔ (Gy 25 ,20 ,15 ,10 ,7.5 ,5 ,0,2.5) واﻟﺘﻰ أﻋﻄﺖ زﯾﺎده ﻓﻰ ﻣﺤﺘﻮى اﻟﺒﺮوﻟﯿﻦ ﺑﺰﯾﺎدة اﻟﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ وﻛﺎﻧﺖ أﻋﻼھ ﺎ ھﻲ 25 ﺟﺮاى ﺣﯿﺚ أﻋﻄﺖ 1,50ﻣﻠﺠﻢ ﺑﺮوﻟﯿﻦ / 100ﺟﺮام وزن ﻃﺎزج. ج- أﺣﺪﺛﺖ اﻟﻤﻌﺎﻣﻠﺔ ﺑﺘﺮﻛﯿﺰات ﻣﻠﻮﺣﺔ (50- 200 ﻣﻞ ﻣﻮل ﻛﻠﻮرﯾﺪ ﺻﻮدﯾﻮم) زﯾﺎدة ﻓﻰ ﻣﺤﺘﻮى اﻟﺒﺮوﺗﯿﻦ وﻛﺎن ﻣﻌﺪل اﻟﺰﯾﺎدة ﻓﻰ ﻣﺤﺘﻮى اﻟﺒﺮوﺗﯿﻦ ﯾﺰﯾﺪ ﺑﺰﯾﺎدة اﻟﻤﻠﻮﺣﺔ اﻟﻰ أن وﺻﻞ اﻟﻰ 2.24 ﻣﻠﺠﻢ / 100ﺟﺮام وزن ﻃﺎزج ﻓﻰ ﺗﺮﻛﯿﺰ 200 ﻣﻞ ﻣﻮل ﻛﻠﻮرﯾﺪ ﺻﻮدﯾﻮم . د- وﺿﺢ ﻣﻦ ﻣﻌﺎﻣﻠﺔ اﻟﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ ﺑﺎﻟﺘﺮﻛﯿﺰات اﻟﻤﻠﺤﯿﺔ (50 ، 100 ، 150، 200 ﻣﻠﻠﻰ ﻣﻮﻟﺮ ) أﻧﮭﺎ زادت ﻣﻦ ﻣﺤﺘﻮى اﻟﺒﺮوﻟﯿﻦ ﺑﺰﯾﺎدة اﻟﺘﺮﻛﯿﺰ ﻣﻦ 1.88:1.55 ﻣﻠﺠﻢ /100 ﻣﻠﻠﻰ وزن رﻃﺐ. ھ - أﻧﺘﺞ اﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﻟﻜﻞ ﻣﻦ اﻟﻤﻌﺎﻣﻼت اﻹﺷﻌﺎﻋﯿﺔ وﺗﺮﻛﯿﺰات ﻛﻠﻮرﯾﺪ ﺻﻮدﯾﻮم إﻟﻰ زﯾﺎدة اﻟﺒﺮوﻟﯿﻦ ﺑﺰﯾﺎدة اﻟﺠﺮﻋﺔ اﻷﺷﻌﺎﻋﯿﺔ وﺗﺮﻛﯿﺰات اﻟﻤﻠﺢ ﻓﺒﯿﻨﻤﺎ أﻋﻄﺖ اﻟﺠﺮﻋﺔ اﻻﺷﻌﺎﻋﯿﺔ10 ﺟﺮاى ﻣﻊ ﺗﺮﻛﯿﺰ ﻣﻠ ﺤﻰ 50 ﻣﻞ ﻣﻮل أﻗﻞ ﻣﺤﺘﻮى ﻣﻦ اﻟﺒﺮوﻟﯿﻦ ﻓﻰ ﺧﻼﯾﺎ اﻟﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ ، ﺑﯿﻨﻤﺎ وﺟﺪ أن اﻟﻤﻌﺎﻣﻠﺔ اﻷﺷﻌﺎﻋﯿﺔ 15 ﺟﺮاى ﻣﻊ ﺗﺮﻛﯿﺰ ﻣﻠﺤﻰ 200 ﻣﻞ ﻣﻮل أﻋﻄﻰ أﻋﻠﻰ ﺗﺮﻛﯿﺰ ﺑﺮوﻟﯿﻦ ﻓﻰ اﻟﺨﻼﯾﺎ ﺣﯿﺚ وﺻﻞ اﻟﻰ 1.08 ﻣﻠﺠﻢ /100 ﻣﻠﻠﻰ وزن رﻃﺐ. و- أﺣﺪث اﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﻟﻠﺠﺮﻋﺎت اﻹﺷﻌﺎﻋﯿﺔ ﻣﻊ ﺗﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ اﻟﻤﺨﺘﻠﻔﺔ زﯾ ﺎدة ﻓﻰ ﻣﺤﺘﻮى اﻟﺨﻼﯾﺎ ﻟﻠﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ ﻣﻦ اﻟﺒﺮوﺗﯿﻦ اﻟﻜﻠﻰ ﻓﻰ ﺑﻌﺾ اﻟﻤﻌﺎﻣﻼت وﺗﻨﺎﻗﺺ ﻓﻰ ﻣﻌﺎﻣﻼت أﺧﺮ ى وﻛﺎن أﻋﻼھﺎ ﻓﻰ اﻟﻤﺤﺘﻮى اﻟﺒﺮوﺗﯿﻨﻰ اﻟﻤﻌﺎﻣﻠﺔ ﺑﺠﺮﻋﺔ اﺷﻌﺎع 10 ﺟﺮاى وﺗﺮﻛﯿﺰ 150 ﻣﻞ ﻣﻮل ﺑﯿﻨﻤﺎ اﻧﺨﻔﺾ اﻟﻤﺤﺘﻮى ﻓﻰ ﺑﺎﻗﻰ اﻟﺠﺮﻋﺎت واﻟﺘﺮﻛﯿﺰات اﻟﻤﺴﺘﺨﺪﻣﺔ. ﺛﺎﻟﺜﺎ:- دراﺳﺔ اﻟﺪﻻﺋﻞ اﻟﻮراﺛﯿﺔ اﻟﺠﺰﯾﺌﯿﮫ ﺑﺎﺳﺘﺨﺪام ﻃﺮﯾﻘﺔ SDS-PAGEوﺟﺪ ﻣﺎ ﯾﻠﻰ:-

2 1- أﺣﺪﺛﺖ اﻟﺠﺮﻋﺎت اﻹﺷﻌﺎﻋﯿﺔ (25 ,20 ,15 ,10 ,7.5 ,5 ,2.5 ,0) ﻣﻦ أﺷﻌﺔ ﺟﺎﻣﺎ ﻇﮭﻮر أو إﺧﺘﻔﺎء ﺣﺰم ﺑﺮوﺗﯿﻨﯿﺔ ﻓﻰ اﻟﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ وذﻟﻚ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﻣﻊ أﻷﺑﺎء اﻟﺜﻨﺎﺋﯿﺔ. 2- أﻋﻄﺖ اﻟﻨﺒﯿﺘﺎت أﻷﺣﺎدﯾﺔ اﻟﻤﺸﻌﻌﺔ إﺧﺘﻔﺎء ﻟﺤﺰﻣﺔ ﺑﺮوﺗﯿﻨﯿﺔ ذات وزن ﺟﺰﺋﯿﻰ ( 44 ﻛﯿﻠﻮ داﻟﺘﻮن) وھﺬا ﯾﻤﻜﻦ اﻋﺘﺒﺎره ﻛﻤﻌﻠﻢ أو دﻟﯿﻞ إﻧﺘﺨﺎﺑﻰ ﺳﺎﻟﺐ ﻟﮭﺬ ه اﻟﻨﺒﺎﺗﺎت اﻟﻤﻌﺎﻣﻠﺔ ﺑﺎﻷﺷﻌﺎع . 3- أﺛﺮت اﻟﻤﻌﺎﻣﻠﺔ ﺑﺎﻟﻤﻠﻮﺣﺔ ﻟﻠﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ ﺗﺄﺛﯿﺮ ا ﻛﻤ ﯿﺎ ووﺻﻔ ﯿﺎ ﻋﻠﻰ اﻟﺤﺰم اﻟﺒﺮوﺗﯿﻨﯿﺔ ﺣﯿﺚ أدت إﻟﻰ إﺧﺘﻔﺎء ﺣﺰﻣﺔ ﺑﺮوﺗﯿﻨﺔ واﺣﺪة ذات وزن ﺟﺰﯾﺌﻰ (63 ﻛﯿﻠﻮ داﻟﺘﻮن ) وھﺬا ﯾﻤﻜﻦ إﻋﺘﺒﺎره ﻛﻤﻌﻠﻢ إﻧﺘﺨﺎﺑﻰ ﺳﺎﻟﺒﻰ ﻟﻠﻨﺒﺎﺗﺎت اﻷﺣﺎدﯾﺔ اﻟﻨﺎﻣﯿﺔ ﺗﺤﺖ اﻟﻈﺮوف اﻟﻤﻠﺤﯿﺔ. 4- دراﺳﺔ اﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﺑﯿﻦ أﺷﻌﺔ ﺟﺎﻣﺎ واﻟﻤﻠﻮﺣﺔ ﻋﻠﻰ ﺑﻌﺾ اﻟﺪﻻﺋﻞ اﻟﻮراﺛﯿﺔ اﻟﺠﺰﺋﯿ ﺔ ﻣﺎ ﯾﻠﻰ: أ- أﺣﺪث اﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﻟﻠﺠﺮﻋﺔ (10 ﺟﺮاى ) ﻣﻊ ﺗﺮﻛﯿﺰات ﻛﻠﻮرﯾﺪ اﻟﺼﻮدﯾﻮم (50 ، 100 ، 200،150 ﻣﻞ ﻣﻮل) ﺗﻐﯿﺮات ﻛﻤﯿﺔ ووﺻﻔﯿﺔ . ﻓﻘﺪ أدت إﻟﻰ اﺧﺘﻔﺎء ﺣﺰﻣﺘﯿﻦ ﺑﺮوﺗﯿﻨﯿﺘﯿﻦ ذات أوزان ﺟﺰﯾﺌﯿﮫ (63، 44 ﻛﯿﻠﻮ داﻟﺘﻮن ). وھﺎﺗﺎن ﯾﻤﻜﻦ إﻋﺘﺒﺎرھﺎ ﻛﻤﻌﻠﻢ إﻧﺘﺨﺎﺑﻰ ﻟﮭﺬه اﻟﻤﻌﺎﻣﻠﺔ . ب- أدى اﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﻟﻠﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ (15 ﺟﺮاى ) ﻣﻊ ﺗﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ (50 ، 100 ، 200،150 ﻣﻠﻠﻰ ﻣﻮﻟﺮ ) إﻟﻰ اﺧﺘﻔﺎء ﺣﺰﻣﺔ ﺑﺮوﺗﯿﻨﯿﺔ ذات وزن ﺟﺰﯾﺌﻲ (63ﻛﯿﻠﻮ داﻟﺘﻮن) وﺗﻌﺘﺒﺮ ﻛﻤﻌﻠﻢ اﻧﺘﺨﺎﺑﻰ ﺳﺎﻟﺐ ﻟﮭﺬه اﻟﻤﻌﺎﻣﻠﺔ . ج – أدى اﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﻟﻠﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ (20 ﺟﺮاى ) ﻣﻊ ﺗﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ (50 ، 100 ﻣﻠﻠﻰ ﻣﻮﻟﺮ) إﻟﻰ ﻇﮭﻮر ﺣﺰﻣﺘﯿﻦ ﺑﺮوﺗﯿﻨﯿ ﺘﯿﻦ ذات أوزان ﺟﺰﯾﺌﯿﺔ (85،109 ﻛﯿﻠﻮ داﻟﺘﻮن ) واﺧﺘﻔﺎء ﺣﺰﻣﺔ ﺑﺮوﺗﯿﻨﯿﺔ ذات وزن ﺟﺰﯾﺌﻲ (63 ﻛﯿﻠﻮ داﻟﺘﻮن ). وھﺬه ﺗﻌﺘﺒﺮ ﻛﻤﻌﻠﻤﺎت إﻧﺘﺨﺎﺑﯿﺔ ﻟﮭﺬه اﻟﻨﺒﯿﺘﺎت أﻷﺣﺎدﯾﺔ اﻟﻤﻌﺎﻣﻠﺔ . د - أﺣﺪث اﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﻟﻠﺠﺮﻋﺔ اﻹﺷﻌﺎﻋﯿﺔ (25 ﺟﺮاى) ﻣﻊ ﺗﺮﻛﯿﺰ اﻟﻤﻠﻮﺣﺔ (50 ﻣﻠﻠﻰ ﻣﻮﻟﺮ) ﻇﮭﻮرﺛﻼﺛﺔ ﺣﺰم ﺑﺮوﺗﯿﻨﯿﺔ ذات اوزان ﺟﺰﯾﺌﯿﺔ (109،61،21ﻛﯿﻠﻮ داﻟﺘﻮن) واﺧﺘﻔﺎء ﺣﺰﻣﺔ ﺑﺮوﺗﯿﻨﯿﺔ ذات وزن ﺟﺰﯾﺌﻲ (63 ﻛﯿﻠﻮ داﻟﺘﻮن). وھﺬه ﺗﻌﺘﺒﺮ ﻛﻤﻌﻠﻤﺎت إﻧﺘﺨﺎﺑﯿﺔ ﻟﮭﺬه اﻟﻨﺒﯿﺘﺎت أﻷﺣﺎدﯾﺔ اﻟﻤﻌﺎﻣﻠﺔ . راﺑﻌﺎ- ﺗﺤﺪﯾﺪ اﻟﺒﺼﻤﺔ اﻟﻮراﺛﯿﺔ اﻟﺠﺰﯾﺌﯿﺔ ﺑﺎﺳﺘﺨﺪام ﺗﻘﻨﯿﺔ اﻟـ RAPD – PCR:- ﺗﻢ ﺗﺤﺪﯾﺪ اﻟﺒﺼﻤﺔ اﻟﻮراﺛﯿﺔ اﻟﺠﺰﯾﺌﯿﺔ وإﯾﺠﺎد ﻛﺸﺎﻓﺎت ﺟﺰﯾﺌﯿﺔ ﻣﺴﺎﻋﺪة ﻟﻼﻧﺘﺨﺎب ﺑﺎﺳﺘﺨﺪام ﺗﻘﻨﯿﺔ اﻟـ RAPD – PCR وﻋﺪد 5 ﻣﻦ اﻟﺒﺎدﺋﺎت اﻟﻌﺸﻮاﺋﯿﺔ واﻟﺘﻰ أﻋﻄﺖ اﻟﻨﺘﺎﺋﺞ اﻟﺘﺎﻟﯿﺔ :- 1- أﺣﺪث اﻟﺒﺎدئ OP-L12 & OP-C13 اﺧﺘﻔﺎء ﺣﺰﻣﺔ ﺑﻮزن ﺟﺰﯾﺌﻰ (bp 930&1140) وﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻰ ﻟﻤﻌﺎﻣﻼت اﻻﺷﻌﺎع وﺗﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ واﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﺑﯿﻨﮭﻤﺎ. 2 - أوﺿﺢ اﻟﺒﺎدئ OP-D07 ﻇﮭﻮر ﺣﺰﻣﺔ ﺑﻮزن ﺟﺰﯾﺌﻰ (bp 980) واﻟﺘﻰ ﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻰ ﻟﻤﻌﺎﻣﻼت اﻻﺷﻌﺎع. 3- أﻇﮭﺮ اﻟﺒﺎدئ OP-L15 ﺣﺰﻣﺔ ﺑﻮزن ﺟﺰﯾﺌﻰ (bp 1120) وﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻰ ﻟﺘﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ.

3 4- أوﺿﺢ اﻟﺒﺎدئ OP-M20 ﺣﺰم ﺑﻮزن ﺟﺰﯾﺌﻰ (bp800، 525 ) واﻟﺘﻰ ﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻰ ﻟﺠﺮﻋﺎت اﻻﺷﻌﺎع وﺗﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ واﻟﺘﺄﺛﯿﺮ اﻟﻤﺸﺘﺮك ﺑﯿﻨﮭﻤﺎ. وأﻇﮭﺮ ﺣﺰﻣﺔ ﺑﻮزن ﺟﺰﯾﺌﻰ (bp 290) وﺗﻌﺘﺒﺮ ﻛﺸﺎف ﺟﺰﯾﺌﻰ ﻟﺘﺮﻛﯿﺰات اﻟﻤﻠﻮﺣﺔ.

4

ﺩﺭﺍﺴﺎﺕ ﻭﺭﺍﺜﻴﺔ ﻋﻠﻰ ﺍﻨﺘﺎﺝ ﺍﻟﺤﺎﻟﺔ ﺍﻷﺤﺎﺩﻴﺔ ﻓﻲ ﺒﻌﺽ ﻨﺒﺎﺘﺎﺕ ﺍﻟﺯﻴﻨﺔ

ﺭﺴﺎﻟﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ﻤﺤﻤﺩﻋﺩﻟﻰ ﻤﺤﻤﺩ ﻤﺼﻁﻔﻲ ﺍﻻﺠﺎﺯﺓ ﺍﻟﻌﺎﻟﻴﺔ (ﺍﻟﺒﻜﺎﻟﻭﺭﻴﻭﺱ) ﻓﻲ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻴﺔ (ﻭﺭﺍﺜﺔ) ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ 1996 ﺍﻟﺘﺨﺼﺹ (ﺍﻟﻤﺎﺠﺴﺘﻴﺭ) ﻓﻰ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻴﺔ (ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﻭﺭﺍﺜﺔ) ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ 2002

ﺍﺴﺘﻴﻔﺎﺀ ﻟﻤﺘﻁﻠﺒﺎﺕ ﺍﻟﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺍﻟﺘﺨﺼﺹ (ﺩﻜﺘﻭﺭﺍﻩ ﺍﻟﻔﻠﺴﻔﺔ) ﻓﻰ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻴﺔ (ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﻭﺭﺍﺜﺔ)

ﻗﺴﻡ ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ ﻜﻠﻴﺔ ﺍﻟﺯﺭﺍﻋﺔ ﺒﺎﻟﻘﺎﻫﺭﺓ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ

1434ﻫـ 2013ﻡ ﺩﺭﺍﺴﺎﺕ ﻭﺭﺍﺜﻴﺔ ﻋﻠﻰ ﺍﻨﺘﺎﺝ ﺍﻟﺤﺎﻟﺔ ﺍﻷﺤﺎﺩﻴﺔ ﻓﻰ ﺒﻌﺽ ﻨﺒﺎﺘﺎﺕ ﺍﻟﺯﻴﻨﺔ

ﺭﺴﺎﻟﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ﻤﺤﻤﺩﻋﺩﻟﻰ ﻤﺤﻤﺩ ﻤﺼﻁﻔﻲ ﺍﻹﺠﺎﺯﺓ ﺍﻟﻌﺎﻟﻴﺔ (ﺍﻟﺒﻜﺎﻟﻭﺭﻴﻭﺱ) ﻓﻲ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻴﺔ (ﻭﺭﺍﺜﺔ) ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ 1996 ﺍﻟﺘﺨﺼﺹ (ﺍﻟﻤﺎﺠﺴﺘﻴﺭ) ﻓﻰ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻴﺔ (ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﻭﺭﺍﺜﺔ) ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ 2002

ﺍﺴﺘﻴﻔﺎﺀ ﻟﻤﺘﻁﻠﺒﺎﺕ ﺍﻟﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺍﻟﺘﺨﺼﺹ (ﺩﻜﺘﻭﺭﺍﻩ ﺍﻟﻔﻠﺴﻔﺔ) ﻓﻲ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻴﺔ (ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﻭﺭﺍﺜﺔ)

ﻗﺴﻡ ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ ﻜﻠﻴﺔ ﺍﻟﺯﺭﺍﻋﺔ ﺒﺎﻟﻘﺎﻫﺭﺓ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ

1434ﻫـ 2013ﻡ ﺃﺠﺎﺯﻫﺎ: ﺃ.ﺩ/ ﻋﺼﺎﻡ ﺃﺤﻤﺩ ﺴﻴﺩ ﺭﺠﺏ ﺃﺴﺘﺎﺫ ﺒﺎﻟﻤﺭﻜﺯ ﺍﻟﻘﻭﻤﻰ ﻟﺒﺤﻭﺙ ﻭﺘﻜﻨﻭﻟﻭﺠﻴﺎ ﺍﻻﺸﻌﺎﻉ – ﻫﻴﺌﺔ ﺍﻟﻁﺎﻗﺔ ﺍﻟﺫﺭﻴﺔ. ﺃ.ﺩ/ ﻣﺤﻤﺪ ﻋﻠﻰ ﻋﺒﺪ اﻟﺮﺣﻤﻦ ﺃﺴﺘﺎﺫ ﺍﻟﻭﺭﺍﺜﺔ – ﻗﺴﻡ ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﻜﻠﻴﺔ ﺍﻟﺯﺭﺍﻋﺔ ﺒﺎﻟﻘﺎﻫﺭﺓ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ ﺃ.ﺩ/ ﻋﺒﺩ ﺍﻟﻬﺎﺩﻱ ﺇﺒﺭﺍﻫﻴﻡ ﺤﺴﻥ ﺴﻴﺩ ﺃﺴﺘﺎﺫ ﺍﻟﻭﺭﺍﺜﺔ – ﻗﺴﻡ ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﻜﻠﻴﺔ ﺍﻟﺯﺭﺍﻋﺔ ﺒﺎﻟﻘﺎﻫﺭﺓ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ

ﺘﺎﺭﻴﺦ ﺍﻟﻤﻨﺎﻗﺸﺔ: / /2013ﻡ

ﺩﺭﺍﺴﺎﺕ ﻭﺭﺍﺜﻴﺔ ﻋﻠﻰ ﺍﻨﺘﺎﺝ ﺍﻟﺤﺎﻟﺔ ﺍﻷﺤﺎﺩﻴﺔ ﻓﻰ ﺒﻌﺽ ﻨﺒﺎﺘﺎﺕ ﺍﻟﺯﻴﻨﺔ

ﺭﺴﺎﻟﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ﻤﺤﻤﺩﻋﺩﻟﻰ ﻤﺤﻤﺩ ﻤﺼﻁﻔﻲ ﺍﻹﺠﺎﺯﺓ ﺍﻟﻌﺎﻟﻴﺔ (ﺍﻟﺒﻜﺎﻟﻭﺭﻴﻭﺱ) ﻓﻲ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻴﺔ (ﺍﻟﻭﺭﺍﺜﺔ) ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ 1996 ﺍﻟﺘﺨﺼﺹ (ﺍﻟﻤﺎﺠﺴﺘﻴﺭ) ﻓﻲ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻴﺔ (ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﺍﻟﻭﺭﺍﺜﺔ) ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ 2002

ﺍﺴﺘﻴﻔﺎﺀ ﻟﻤﺘﻁﻠﺒﺎﺕ ﺍﻟﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺍﻟﺘﺨﺼﺹ (ﺩﻜﺘﻭﺭﺍﻩ ﺍﻟﻔﻠﺴﻔﺔ) ﻓﻲ ﺍﻟﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻴﺔ (ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﺍﻟﻭﺭﺍﺜﺔ)

ﻗﺴﻡ ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ ﻜﻠﻴﺔ ﺍﻟﺯﺭﺍﻋﺔ ﺒﺎﻟﻘﺎﻫﺭﺓ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ

1434ﻫـ 2013ﻡ ﻟﺠﻨﺔ ﺍﻹﺸﺭﺍﻑ: ﺃ.ﺩ/ ﻋﺒﺩ ﺍﻟﻬﺎﺩﻱ ﺇﺒﺭﺍﻫﻴﻡ ﺤﺴﻥ ﺴﻴﺩ ﺃﺴﺘﺎﺫ ﺍﻟﻭﺭﺍﺜﺔ – ﻗﺴﻡ ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﻜﻠﻴﺔ ﺍﻟﺯﺭﺍﻋﺔ ﺒﺎﻟﻘﺎﻫﺭﺓ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ ﺍﻟﻤﺭﺤﻭﻡ ﺃ.ﺩ/ ﻋﺒﺩﺍﻟﻌﺯﻴﺯ ﺤﺴﻥ ﻋﺒﺩ ﺍﻟﻠﺔ ﺃﺴﺘﺎﺫ ﺍﻟﻭﺭﺍﺜﺔ – ﻗﺴﻡ ﺍﻟﻨﺒﺎﺕ ﺍﻟﺯﺭﺍﻋﻲ – ﻜﻠﻴﺔ ﺍﻟﺯﺭﺍﻋﺔ ﺒﺎﻟﻘﺎﻫﺭﺓ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ ﺃ ﺩ/ ﺃﻴﻤﻥ ﻋﺒﺩ ﺍﻟﻤﺠﻴﺩ ﺍﻟﻔﻘﻰ ﺃﺴﺘﺎﺫ ﺍﻟﺒﻴﻭﺘﻜﻨﻭﻟﻭﺠﻰ – ﺍﻟﻤﺭﻜﺯ ﺍﻟﻘﻭﻤﻰ ﻟﺒﺤﻭﺙ ﻭﺘﻜﻨﻭﻟﻭﺠﻴﺎ ﺍﻻﺸﻌﺎﻉ – ﻫﻴﺌﺔ ﺍﻟﻁﺎﻗﺔ ﺍﻟﺫﺭﻴﺔ.