Shoot Tip Culture Research Articles

In vitro Secondary Metabolite Production from the Roots of Decalepis arayalpathra KMA 05 Clones and its Antimicrobial Potential using Methylobacterium sp. VP103 as an Elicitor

Decalepis arayalpathra KMA 05 clones were elicited with a bacterium, Methylobacterium sp. VP103 isolated from the leaves of ex-vitro established plantlets of D. arayalpathra KMA 05 clones, with the aim of increasing the production of root specific secondary metabolites. D. arayalpathra KMA 05 shoot tip cultures were triggered for root initiation by keeping the cultures for one month in ½ strength Woody Plant Medium (WPM)+Thidiazuron (TDZ) 0.3 mg/l. They were then shifted to ½ strength WPM supplemented with auxins [0.5 mg/l indole-3-butyric acid (IBA) + 0.05 mg/l Naphthalene acetic acid (NAA)] along with 40 mg/l activated charcoal with a fixed concentration (0.4 mg/l) of lyophilized bacterium powder (LBP) dissolved in 30% methanol. The effect of elicitation was studied on three root specific secondary metabolites, i.e. 2-hydroxy 4-methoxy benzaldehyde (MBALD), 2-methoxy-4-vinylphenol (2M4VP) and α-amyrin. The production of α-amyrin was found to be enhanced in all 12, 24 and 36 week old treated root extracts. Antibacterial activity was also found to be enhanced against all the bacteria selected from the 36 week elicited extracts among which Rhodococcus sp. UKS7 (34 ± 0.0 mm.) showed maximum inhibition. The findings provide an insight into the use of Methylobacterium sp. VP103 as an elicitor in the growth of the roots and enhanced production of α-amyrin.

Elimination of Grapevine rupestris stem pitting-associated virus from Vitis vinifera ‘Kyoho’ by an antiviral agent combined with shoot tip culture

Abstract We evaluated the ability of different concentrations of Ribavirin (15 and 25 μg/mL, R15 and R25) to eliminate Grapevine rupestris stem pitting-associated virus (GRSPaV) from in vitro-cultured grapevine plants. Vitis vinifera ‘Kyoho’ plants infected by GRSPaV were used as the original plant materials. All of the plants survived in the treatment with R15 and R25 for 85 days, but all Ribavirin-treated plants exhibited phytotoxicity symptoms after 25 days. To ensure the accuracy of detection, optimal primer pair combinations (cpF1R2-cpF2R1 and rep1F1R2-rep1F2R1) were used to assess the eradication efficiency of GRSPaV from regenerated grapevine plants with a nested reverse transcription polymerase chain reaction. The results showed that the efficiency of the GRSPaV eradication was associated to virus titer of regenerated plant in different detection period. All surviving regenerated plants were tested for two rounds and the average elimination rates of GRSPaV in the two periods showed more different, 74.8% (113/151) and 52.3% (79/151), respectively. In addition, the efficiency of the GRSPaV eradication also depended on the concentrations and treatment duration of Ribavirin. The average elimination rates for the regenerated R25-treated and R15-treated plants in the second round detection were 54.1 and 50.6%, respectively. Moreover, the elimination rates for the R25-treated plants were 21.4, 37.5, and 77.8% after 45, 65, and 85 days of treatment, respectively. Combination of shoot tip culture with chemotherapy may greatly increase the efficiency of viral eradications.

Micropropagation of thyme plant (Thymus vulgaris)

The current research was conducted at Vegetable and Floriculture Department in the experimental station and tissue culture laboratory, Agriculture Faculty, Mansoura University. The objective of this research was to develop a direct plant regeneration of a valuable aromatic and medicinal plant (Thymus vulgaris) by in vitro culture of shoot tips or nodal segments from mature plants on MS medium supplemented with different cytokinin type at different concentrations. Nodal segments were found to be more efficient than shoot tips for Thymus vulgaris shoots regeneration on MS medium (Murashig and Skoog 1962) supplemented with 6-benzyladenine (BA) at 2 mg/L. The best elongation was obtained on MS medium supplemented with BA at 2 mg/L in combination with GA3 at 0.5 mg/L.The best rooting of shoots was obtained on MS medium augmented with α-naphthaleneacetic acid (NAA) at 1.5 mg/L. Regenerated plants were successfully established in pots filled with mixture of soil: peat moss (1: 1 v/v). In vitro Thymus vulgaris plants had a survival rate of 100 %, and showed healthy and uniform growth.

Patateste (Solanum tuberosum L.) in vitro Mikrotuberizasyon Üzerine Fotoperiyot, Jasmonik Asit ve Aktif Kömürün Etkileri

In this study, effects of added agar, jasmonic acid (JA), activate charcoal (AC) with photoperiod regime on in vitro microtuber formation in Solanum tuberosum cv. Marfona. Study has actualized as 4 main steps; shoot formation of the tubers of S. tuberosum, in vitro shoot-tip culture, micropropagation and microtuberization. The effects of JA (0.0-0.2 mgL-1), agar (0.5% or 0.7%), AC (0 and 0.2%) microtuberization were investigated. The highest microtuber yield (137%) and the highest microtuber average (183 mg/tuber) were obtained at the double-layer Murashige and Skoog (MS) medium which contains 0.2% AC, 0.5% agar under darkness conditions. It has been determined that positive effect of AC on microtuber formation is higher than JA. AC was found much more effective in the dark.

English

Taro leaf blight disease has recently been reported in Cameroon to cause between 50 and 100% yields loss of taro in most of the agro-ecological crop growing regions. This has led to a significant reduction in disease-free planting materials, edible crop and increased. The Meristem culture technique has been used to produce crop plants free of viruses and fungi especially in vegetative propagated colocassia plants. This aimed at applying in-vitro micro-propagation technique for sustainable production of four local taro cultivars in Cameroon. This study was conducted at the Root and Tuber Tissue Culture Laboratory, of the Institute of Agricultural Research for Development (IRAD), Bambui from April 2015 to November 2016. Micro-plants from four local taro cultivars were produced in vitro from apical meristem tips. The tip meristems were excised from corms of the four local taro cultivars. The excised explants were surface sterilized with alcohol and sodium hypochlorite in sequence steps at different concentrations. Meristems were cultured at establishment stage on Murashige and Skoog (MS) medium with 30 g of sugar, 1.1 ml of 6-benzylaminopurine and 7 g of agar. Shoots proliferation was induced in MS with 2.2 ml of 6-benzylaminopurine (BAP). Result shows a significant difference at p≤0.5 in number of shoots, petiole length, open leaf and corm diameter among the cultivars and no significant variation in mean number of senescence leaf with respect to all the cultivars, at 60 days of shoot tip culture. At rooting stage, taro shoots were cultured on MS media supplemented with 10 ml of 0.1 mg/ml naphthalene acetic-acid (NAA). Roots were produced on all the cultivars with excellent mean growth rate of 14.7 ± 0.69 recorded in cultivar with dark green petiole and small leaves.   Key words: Taro cultivars, in-vitro micro-propagation, benzylaminopurine, naphthalene acetic-acid, Murashige and Skoog.

Biotechnology for Conservation and Utilization of Agricultural Plant Genetic Resources in Nepal

Agricultural biodiversity is the basis of human life and food security. Nepal with 577 cultivated species possesses huge diversity at varietal as well as landrace levels. In most agricultural crops the rapid genetic erosion due to several reasons is a common phenomenon. Thus, considering the importance of agricultural biodiversity declared by Convention on Biological Diversity for sustainable food production, National Agriculture Genetic Resources Center (NAGRC) has been established for conservation and sustainable utilization of agricultural biodiversity. This paper thus delineates the application of biotechnological tools adopted by NAGRC for effective and efficient conservation and use of agricultural plant genetic resources (APGRs). Among the adopted technologies, tissue bank using shoot tip culture of vegetatively propagating and recalcitrant crops eg potato, sugarcane, banana, sweet potato, etc are in function. Under the molecular marker technology, currently random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers have been used for developing DNA profiles, identifying duplicates in the collections, assessing genetic diversity and screening accessions against economic traits. DNA bank has also been created for storing DNA of indigenous crops and these DNA can be accessed for research and study. Genotypic database has been developed for chayote, finger millet, wheat and maize for identification and selection of the accessions.Journal of Nepal Agricultural Research Council Vol.3 2017: 49-59

English

Stevia rebaudiana Bert. is a non-caloric sweetener plant of medicinal and commercial values. This study was performed to micropropagate Stevia and improve its ability to withstand the sudden shocks of the environmental changes during acclimatization by using endomycorrhizae and/or Pseudomonas fluorescens. In vitro establishment of Stevia was superior by culturing shoot tips and nodal segments of mature plants on Murashige and Skoog medium supplemented with 0.5 mg/l 6-benzyl adenine and 1 mg/l β-naphthalene acetic acid, and nodal segments gave better response when compared to shoot tips. While 6-benzyl adenine alone at 1 mg/l was found to be the most promising concentration for shoots multiplication for four successive subculturing. The best rooting response was obtained on half strength Murashige and Skoog medium supplemented with 1 mg/l β-naphthalene acetic acid. Adding 8 ml/l mycorrhizal root extract gave 100% rooting with the highest mean number and length of roots and shoot height. Inoculation of plantlets with endomycorrhizal spores alone in non-sterilized soil increased the survival percentage to 90% and gave the highest mean number and length of both shoots and roots, as well as leaves number. Moreover, they recorded the highest values of total chlorophyll, NPK and relative water contents. Key words: Stevia, micropropagation, mycorrhizae, mycorrhization helper bacteria.

Agronomic performance of cassava genotypes from the in vitro shoot tip culture submitted to clonal cleaning

Agronomic performance of cassava genotypes from the in vitro shoot tip culture submitted to clonal cleaning

Cleaning cassava genotypes infected with cassava frogskin disease via in vitro shoot tip culture

This study aimed to develop a methodology for eliminating cassava frogskin disease (CFSD) from in vitro shoot tip culture by associating thermotherapy and tetracycline. Cuttings from different accessions (BGM0232, BGM0315, BGM0464, BGM584, BGM0841, and BGM1342), infected with CFSD according to visual inspection of the disease symptoms, were used for cleaning. To verify the absence of other diseases, the plants were indexed for Cassava common mosaic virus - CsCMV (by ELISA) and Cassava vein mosaic virus - CsVMV (by polymerase chain reaction, PCR), proving that the accessions were free of these viruses, except for BGM0315 and BGM0464, which were infected with CsVMV. Subsequently, the cuttings were submitted to different tetracycline concentrations for 3 min, and then subjected to thermotherapy under different temperatures (35°, 38°, 40°, 45°, and 55°C). Shoots of 2 cm were harvested, and their surfaces were sterilized in a laminar flow chamber. Subsequently, the shoot tips of different sizes were removed (0.2, 0.4, 0.5, and 1.0 mm) for inoculation in a culture medium with tetracycline at the same concentrations in which the cuttings were dipped. After 60 days of cultivation, the explants were transferred to a multiplication medium without antibiotics. Thirty days after the transfer, the viability of the regenerated plants was evaluated, which were then acclimatized for 70 days in a greenhouse and transferred to the field. After 7 months, a visual analysis of the symptomatic roots and a PCR analysis were held to prove the elimination of CFSD and CsVMV from the accessions infected with these viruses (BGM0315 and BGM0464), respectively. Most of the treatments resulted in 100% cleaning of CFSD-infected plants. From accessions that were also infected with CsVMV, only 2% of the plants remained infected, also demonstrating the cleaning efficiency of this protocol for this disease.

English

Enset is an important food crop produced in Ethiopia with great role in food security. The demand of the crop is increasing throughout the country. However, the production as a whole is decreasing due to devastation by enset bacterial wilt. The studies were conducted to develop the procedure for obtaining multiple disease free plantlets from infected enset plants. Shoot tip explants from infected suckers of three clones Arkiya, Digomerza and Mazia were cultured on MS media supplemented with different combinations of benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) each with concentration of 0, 1.5, 3, 4.5 and 6 mg/L with 0, 0.5, 1, 1.5 and 2 mg/L, respectively. The effect of growth regulators on shoot growth parameters was examined. The minimum days (11.66) for multiple shoot induction were recorded for Mazia on media with 4.5 mg/L BAP and 1.5 m/L NAA. The maximum number of shoots (23.0) was also obtained for Mazia on the same hormone combination as for days of induction. Whereas, the maximum shoot length (8.1 cm) was recorded for Digomerza on media with 3 and 1 mg/L NAA. Similarly, for root induction and growth MS media with different concentrations of indulebutyric acid (IBA; 0, 0.5, 1, 1.5 and 2 mg/L) were evaluated. The minimum days (10.5) to root induction was observed for Mazia on media with 1.5 mg/L IBA and the maximum root number (3.8) was recorded at 2 mg/L IBA. In the efficiency of shoot tip culture for Xanthomonas pathogen elimination, sample suspension was prepared from shoots regenerated from diseased suckers and transferred on semi-selective yeast peptone sucrose agar (YPSA) medium. The result of colony observation indicated that many microbes are living in enset saprophytically as mixed colony growth was observed within 24 h after sample culturing. Pathogenesity test on clean suckers of susceptible clone showed that the colonies grown were due to endophytic microbes since none of the colonies were capable to develop disease symptoms as sample of the pathogen strain. Key words: Ensete ventricosum, benzylaminopurine (BAP), indulebutyric acid (IBA), naphthaleneacetic acid (NAA), shoot tip, Xanthomonas, yeast peptone sucrose agar (YPSA).

Elimination ofApple stem grooving virusfrom ‘Mansoo’ pear (Pyrus pyrifoliaL.) by an antiviral agent combined with shoot tip culture

Elimination of<i>Apple stem grooving virus</i>from ‘Mansoo’ pear (<i>Pyrus pyrifolia</i>L.) by an antiviral agent combined with shoot tip culture

Ultrastructure study of Vanda Kasem's Delight orchid's protocorm-like body

ABSTRACT Growing orchids has been classified as an international business since it covers 8% of the world floriculture trade. Thus, large-scale micropropagation of orchid using tissue culture techniques and improvement of some essential traits, such as resistances to various diseases and pests, and tolerances to environmental stresses, such as low temperatures and low light intensities, via genetic engineering acknowledged the orchids as one of the top ten cut flowers. Protocorm-like bodies (PLBs) are excellent explants for clonal propagation, artificial seeds development and genetic engineering since they are organized and easily regenerable somatic embryos that propagate rapidly. Vanda Kasem's Delight hybrid orchid's PLBs derived from shoot tip culture were examined in order to understand the fundamental ultrastructure of PLB. Examination of the cross sections of PLB revealed that PLB is made of a discrete bipolar structure that contains anterior and posterior meristems. Actively dividing meristem cells and outer layer lined by several rows of small and isodiametric cells with a dense cytoplasm and a prominent nucleus were also observed via cytological studies. Scanning Electron Microscope investigations revealed that the surface of PLBs is occupied by vertically positioned branched trichome appendages and regularly spaced stomatal openings with two guard cells. Organelles such as mitochondria of various sizes, shapes and biconvex chloroplast in the cytoplasm were revealed by Transmission Electron Microscope analysis. Hence, inheriting actively dividing cells, and bestowed with elements related to transpiration, photosynthesis and energy synthesizing power house makes PLB a suitable explant for micropropagation and genetic engineering studies.

Pengaruh Kinetin dan BAP terhadap Pertumbuhan dan Perkembangan Embrio Somatik Tanaman Sagu (Metroxylon sagu Rottb.)

&lt;p&gt;Effect of Kinetin and BAP to Growth and Development of&lt;br /&gt;Somatic Embryos of Sago Palm (Metroxylon sagu Rottb.).&lt;br /&gt;Imron Riyadi. Somatic embryos induction in sago palm&lt;br /&gt;(Metroxylon sagu Rottb.) have succesfully developed. Kinds&lt;br /&gt;and concentration of plant growth regulators (PGR’s)&lt;br /&gt;influence to growth and development of somatic embryos.&lt;br /&gt;The research was conducted to determine the optimal concentration&lt;br /&gt;of BAP and kinetin for proliferation, maturation&lt;br /&gt;and germination of sago palm somatic embryos. Cotyledonstage&lt;br /&gt;of somatic embryo derived from shoot tip cultures&lt;br /&gt;cultured on Modified Murashige-Skoog (MMS) with halfstrength&lt;br /&gt;macro-salts and added with 30 g/l sucrose, 2 g/l&lt;br /&gt;gelrite, 1 g/l activated charcoal. pH of media was adjusted at&lt;br /&gt;5.6 before sterilized. The media were supplemented with&lt;br /&gt;0.1-2.0 mg/l BAP and 0.1-2.0 mg/l kinetin in combination with&lt;br /&gt;0.01 mg/l ABA each for supporting growth and development.&lt;br /&gt;The cultures was incubated at 26+1oC under a 12-h&lt;br /&gt;photoperiod with lighting providing an intensity 20 μmoles&lt;br /&gt;photons/m2/second for 11 weeks with replication 10 times.&lt;br /&gt;The results showed that the highest of somatic embryo&lt;br /&gt;proliferation was achieved in a culture medium with BAP at&lt;br /&gt;0.5 mg/l + 0.01 mg/l ABA with an expression rate of 94%, the&lt;br /&gt;best maturation at 1.0 mg/l kinetin + 0.01 mg/l ABA with an&lt;br /&gt;expression rate of 93.5% and the most germination at 2.0&lt;br /&gt;mg/l kinetin + 0.01 mg/l ABA with an expression rate of&lt;br /&gt;100%. Transfer of these germinants to gelled media without&lt;br /&gt;PGR’s led to the development of normal plantlets.&lt;/p&gt;

Water and Chlorophyll Content and Leaf Anatomy of Patchouli Planlet (Pogostemon cablin Benth.) Resulted by Shoot-tip Culture Experience Hyperhydricity after Treatment of Modification Ammonium nitrate or Macro salt Concentration on MS medium (Murashige Skoog)

Hyperhydricity is a symptom of abnormal morphological and physiological function which inhibits the regeneration of plantlets. In general, the main symptom of hyperhydricity is a change in the condition of the plantlets which looks clear (Glassy) as a result of low levels of chlorophyll, the high water content in the plantlets, and the abnormal anatomical structure of the leaves. Hyperhydricity can be controlled by reducing cytokinin concentration, increasing gelling agent concentration, and reducing ammonium nitrate and macro salt concentration on medium. Objective of this research was to reduce hyperhydricity in shoot tip culture of patchouli by modification of ammonium nitrate and macro salt concentration on MS medium. The various treatment concentrations of ammonium nitrate were 0 mg.L -1 (0), 41.25 mg.L -1 (¼ concentration), 825 mg.L -1 (½ concentration), 1650 mg.L -1 (1 concentration) and macro salt MS with 0, ¼ MS, ½ MS, MS with 5 replications. Hyperhydricity on patchouli shoots could be lowered, as indicated by the decrease in water content from 96% to 90-91%, the increase in total chlorophyll content, and the increased number of palisade cells and stomata on the leaf treatment outcome. The concentration treatment of ammonium nitrate showed better results than the concentration of macros salt in increasing the total chlorophyll content, but it did not differ significantly in lowering water levels and increasing the number of palisade cells and stomata. ¼x concentration treatment of ammonium nitrate could increase chlorophyll content of 0.16 to 0.97 mg.g -1 , but MS with 1x concentration showed the best result in the increase of number of palisade cells and stomata of the leaves. Keywords: Ammonium nitrate, Hyperhydricity, Macro Salt, Shoot-tip culture.

English

In vitro propagation of banana preferably use sword sucker as explant source where microbial contamination poses a great problem in establishment of aseptic cultures. This study demonstrates up to 36% reduced microbial contamination in aseptic culture establishment and subsequent micropropagation due to osmotic stress induction in the banana suckers. Osmotic stress was induced by keeping the freshly collected suckers in shade and measuring fresh weight at 0, 7, 14, 21, and 28 days interval to ascertain loss of moisture. Stress induced for 21 days showed 58.85% moisture loss showing lowest contamination upto 40% against 76% for fresh suckers. Micropropagation of Musa sp. (Malbhog variety) through shoot tip culture of stressed suckers was carried out in Murashige and Skoog (MS) medium supplemented with 1 to 2 mg/l &nbsp;6-benzyl-aminopurine (BAP), 2 to 4 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l &nbsp;&alpha;-naphthalene acetic acid (NAA). Multiplication of plantlets was observed till 6th passage. From 19 aseptically established explants of 21 days stressed sucker sub cultured in MS medium fortified with 1.0 mg/l BAP and 0.5 mg/l NAA, for 6th passage produced 5122 plantlets. Regenerated micro shoots were rooted in MS medium fortified with 0.5 mg/l IAA. The plantlets were hardened in polybag containing soil and seasoned cow dung. &nbsp; Key words: Osmotic stress, shoot tip culture, in vitro, Musa, Malbhog.

Embryogenic Callus Induction of Pencil Orchid (Papilionanthe hookeriana Rchb.f.) Through in Vitro Culture

Papilionthe hookeriana Rchb.f. (Orchidaceae), popularly known as ‘Anggrek Pinsil’ in Indonesia, is a perennial ephiphyte orchid, found only at Dendam Tak Sudah Lake in Bengkulu. The aims of this research were to find the best sterilization technique of P. hookeriana explant and to induce maximum formation of embryogenic calli. Rapid multiplication of this orchid was achieved through culture of shoot tips and young leaf segments of mature plants by in vitro cultured on Murashige and Skoog (MS) basal medium enriched with 50 g L -1 banana pulp. The experiment used Completely Randomized Design (CRD) with three replications. In the first stage, the explants were sterilized using three compositions of sterilant. In the second stage, the explants were planted on the MS basal medium with addition of five levels of 2,4-Diclhorophenoxy Acetis Acid concentrations, namely 0, 0.25, 0.50, 0.75, and 1.00 mg L -1 . The result showed that the best sterilization method to reduce explant contaminant was method 3, in which the explant was washed with detergent, rinsed with flowing water, soaked in 0.1% (v/v) HgCl 2 solution for 30 minutes, soaked again in the 10% (v/v) Natrium hypocloride solution for 20 minutes, rinsed three times with sterile water before planted and then soaked in sterile water + 10% (v/v) betadhine before planted on treated medium. This method was able to reduce contamination levels up to 70% from explants cultured for 5 months on MS medium. MS medium added with 1.00 mg L -1 2,4-D produced the highest number of embryogenic calli, and the biggest callus diameter ( 3.5 cm ), characterized by transparent green color and friable callus structure

Factors affecting in vitro shoot-tip culture of banana

Various factors affecting in vitro shoot-tip culture of banana were examined to standardize the suitable culture media, as well as the culture environment. Three local cultivars identified for experiments, viz., 'Amritsagar' (AAA), 'Bharatmoni' (AAA) and 'Kanaibasi' (AA), have high potential in terms of plant growth, yield and fruit quality in commercial cultivation. Shoot tips from 3-month-old suckers of these cultivars were selected as explants source and were cultured on modified MS medium containing different concentrations and combinations of auxins and cytokinins, along with some other supplements, and incubated under different temperatures. Highest frequency of explant survival, regrowth and highest multiplication of shoots was observed on modified MS medium with 5 mg L-1 6-benzylaminopurine, 1 mg L-1 indole-3-acetic acid (IAA), 2 g L-1 activated charcoal and 50 mg L-1 casein hydrolysate, at a temperature of 27°C. About 20 shoots per plant could be achieved in this medium within 6-8 weeks of culture. Healthy roots were induced by subculturing shoots on half-strength MS medium supplemented with 2 mg L-1 of indole-3-butyric acid within 15 days of incubation. Regenerated plantlets were transferred to soil after hardening. After attaining the average height of 25 cm, the plants were transferred in the field. The plants showed vigorous growth in terms of total leaves per plant, total leaf area, pseudostem circumference and height of the plants. Preliminary results show that the quality and the productivity of the in vitro-derived plants are superior in comparison to conventional sucker-derived plants.

Callose Deposition in Plasmodesmata and Viroid Invasion of the Shoot Apical Meristem.

Callose Deposition in Plasmodesmata and Viroid Invasion of the Shoot Apical Meristem.

Culture of shoot tips from adventitious shoots can eradicate Apple stem pitting virus but fails in Apple stem grooving virus

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from ‘Gala’ apple using shoot tips from of adventitious shoots derived from leaf segments. Leaf segments were excised from in vitro 4-week-old stock shoots and cultured on shoot regeneration medium containing 9.1 µM thidiazuron and 0.25 µM indole-3-butyric acid (IBA) to induce adventitious shoot formation. Shoot tips of different size and different developmental stage were excised from adventitious shoots and cultured on shoot tip culture medium containing 1.1 µM 6-benzyladenine and 0.05 µM IBA. Results showed size and developmental stage of shoot tips excised from adventitious shoots did not influence survival rate, but significantly affected shoot regrowth rate and ASPV-free frequency. Shoot regrowth rates increased from 10 to 15 % in 0.3 mm shoot tips containing two leaf primordia (LP) excised after 2–3 weeks of shoot regeneration, to 53–55 % in those containing three LP excised after 3–4 weeks. The highest shoot regrowth rate (82 %) was obtained in shoot tips of 0.4 mm shoot tips containing four LP excised after 4 weeks. ASPV-free frequencies (95–100 %) were high in 0.2–0.4 mm shoot tips containing two to three LP excised after 2–4 weeks, but low (20 %) in 0.4 mm shoot tips containing four LP excised after 4 weeks. None of the shoots regenerated from the shoot tips were ASGV-free, regardless of the size and developmental stage at which shoot tips were excised. Histological studies and virus localization provided explanations to the varying frequencies of the virus eradication using different size of shoot tips that were excised from adventitious shoots at different developmental stages. The protocol described here was characteristic of high frequency ASPV eradication and high efficiency of production of shoot tips that were used for virus eradication, and thus may have potential applications to virus eradication in other plant species.

SHORT TERM PRESERVATION FOR FIG (Ficus carica cv. Black fig) BY DIFFERENT OSMOTIC STABILIZERS

Fig (Ficus carica L.) is a deciduous tree that belongs to the Moraceae family, and one of the most suitable species for cultivation in semiarid environments found in regions of the Mediterranean and Middle-East; it is considered to be one of the important crop plants grown in Egypt. Many of the species are currently threatened; continuously vulnerable to loss and genetic transmutation due to the absence of safe longterm preservation. In vitro preservation of vegetative propagated genetic resources aided in providing an effective conservation system for the guarantee of food supplies. The present study used shoot tip cultures that were obtained from the black fig (Ficus carica) from Siwa. Shoot tip explants were cultured on conservation media composed of full strength MS medium with 0.8% (w/v) agar and different concentrations of different osmotic agents. The study investigated the use of osmotic stabilizers (mannitol and sorbitol, with concentration of [40 g/L, 50 g/L and 60 g/L]) in the media at two different temperatures (5C and 10C) through a 3 months period in order to determine which osmotic stabilizer, concentration, and temperature would display an eminent effect on the in vitro short-term storage of fig shoot cultures. A (30 g/L) concentration of sucrose was used as the control media. Results were in favor of sorbitol (50 g/L) at a temperature of 5C. In addition Inter Simple Sequence Repeats (ISSR) marker was performed to assess molecular characterization of genetic identity and stability; results illustrated that no genomic instability and mutations were found in the propagated fig (Ficus carica L.) cultures.