Tamara S. Al-Qudah

In vitro Production of Silymarin from Silybum marianum L.

Silybum marianum L. is a wild medicinal herbal plant in Jordan. It is widely used in folk medicine due to its high content of silymarin. In vitro production of silymarin was experimented at different concentrations (0.0, 0.4, 1.0, 1.6 or 2.0 mg/l) of growth regulators (6-furfurylaminopurine (kinetin), 6-benzylaminopurine (BA), or 6-(gamma,gamma- Dimethylallylamino) purine (2ip)) and different concentrations (15, 30,45,60 mg/l) of carbon sources (glucose, fructose, and sucrose). HPLC (High Performance Liquid Chromatography) analysis was used to identify silymarin components. In vitro grown S. marianum on MS (Murashige and Skoo 1962) medium supplemented with (1.6 mg/l) kinetin and (0.1 mg/l) 1-Naphthaleneacetc acid (NAA) gave the highest silymarin content of (0.84%) silybin and (0.49%) silydanin as compared with cultures grown on hormone-free MS media which contained (0.36%) silybin and (0.30%) silydanin. In vitro grown S. marianum on MS medium supplemented with (2.0 mg/l) BA and 0.1 mg/l NAA yielded (0.67%) silybin and (0.37%) silydanin, while MS media supplemented with (1.0 mg/l) of 2iP gave (0.72%) silybin and (0.24%) silydanin. Otherwise, the in vivo (wild) grown shoots of S. marianum gave (1.07%) for silybin and (0.46%) sildyanin. Among carbon source, glucose at (45 g/l) gave (1.63%) of silymarin content. Results indicated a significant use of in vitro grown cultures for silymarin production.

Cryopreservation and Genetic Stability Assessment of Threatened Medicinal Plant ( Ziziphoratenuior L. ) Grown Wild in Jordan

Ziziphora tenuior L. is one of the important medicinal plants that belong to the Lamiaceae family. It is a rare species with a promising medicinal potential and grows wild in the southern part of Jordan. Unfortunately, this plant might be totally extinct from the wild due to over-exploitation. Two cryopreservation techniques (encapsulation-dehydration and encapsulationvitrification) were applied for in vitro conservation of this valuable medicinal plant, and after that the explants were tested for their genetic stability using Amplified Fragment Length Polymorphism (AFLP) technique. In the encapsulation-dehydration experiment, the results revealed that 40% of the cryopreserved shoot tips survived when they were dehydrated chemically on 0.75 M sucrose in MS supplemented media for one day and exposed to air dehydration for 6 hrs. Moreover, the best recovery rate (20%) was obtained when either 0.5 M or 0.75 M sucrose MS supplemented media were used as preculture media for the shoot tips for one day, followed by air dehydration for 4 or 6 hrs. Meanwhile, in the encapsulation -vitrification experiment, the highest survival (37.5%) and recovery (10%) percentages of the cryopreserved shoot tips were obtained when the encapsulated shoot tips were pretreated for 60 min. with the loading solution before being exposed to PVS2 vitrification solution and LN. AFLP technique had clearly showed that, there were no genetic variations between the shoot tips of Ziziphora tenuior L., before and after cryopreservation.

Cell Suspension and In Vitro Production of Colchicine in Wild Colchicum Hierosolymitanum Feib

Callus was induced from seeds of Colchicum hierosolymitanum Feib inoculated on the surface of MS media supplemented with 0.45 µM 2, 4-dichlorophenoxyacetic acid under dark conditions. Then callus was cultured on MS media supplemented with 4.52 µM 2, 4-dichlorophenoxyacetic for growth and maintenance. Friable callus from the fourth generation was transferred to liquid MS media supplemented with 0.54 µM 1-naphthaleneacetic acid to form cell suspension. Cells were successfully subcultured every 27 days on the same liquid media supplemented with 0.54 µM 1- naphthaleneacetic acid. Higher concentration (9 µM) of 6-benzyladenine with 0.45 µM 2, 4-dichlorophenoxyacetic acid resulted in higher cells fresh weight, while 1-naphthaleneacetic acid combinations with 6-benzyladenine had no effect on cell growth. On the other hand, the time for subculturing the cells into new fresh liquid media was determined to be after 27 days of incubation. (-) -Colchicine was identified in callus and cell suspension of C. hierosolymitanum by performing HPLC analysis against standard. Different ratios of NH +4 : NO -3 were used to study their effect on (-)-colchicine content, the highest colchicine content of 0.070 mg g -1 DW was obtained at 30 mM NH 4 + of total nitrogen. Colchicine alkaloid was highest, 0.090 mg g -1 DW, at 0.1 M of sucrose after 4 weeks incubation. (-) - Colchicine alkaloid was not detected in callus grown on sucrose free media. Maximum production of colchicine, 0.235 mg g -1 DW, was obtained in callus extracts of 60 days old callus grown under dark conditions. Cell suspension had 0.012 mg g-1 DW (-) -colchicine from suspended cells grown under dark. (-) -Colchicine content of callus incubated under dark (0.095 mg g -1 DW) was higher than light (0.070 mg g -1 DW) condition.

In Vitro Preservation of Transgenic Tomato (Solanum lycopersicum L.) Plants Overexpressing the Stress-Related SlAREB1 Transcription Factor

In vitro preservation of transgenic tomato lines overexpressing the stress-responsive transcription factor SlAREB1 was studied by using slow growth and cryopreservation techniques. Slow growth preservation was performed by using different concentrations of sucrose (0, 100, 200, 300 mm) and abscisic acid (0, 4, 8, 12 μm) in Murashige and Skoog (MS) media, while cryopreservation was conducted by using encapsulation dehydration, V-cryoplates and seeds. Significant differences were observed between tested lines grown on MS media supplemented with 200 mm sucrose where transgenic lines overexpressing SlAREB1 showed improved growth when compared with negative control. The addition of abscisic acid (ABA) to the preservation media affected negatively transgenic lines growth and development when compared with ABA-free media. In encapsulation dehydration, non-cryopreserved transgenic lines overexpressing SlAREB1 pretreated in 0.8 M sucrose for 1 day and subjected to different dehydration periods showed significantly higher survival percentages when compared with negative control. For V-cryoplates technique, cryopreserved transgenic lines overexpressing SlAREB1 treated in 0.3 M sucrose for 3 days with or without cold acclimatization showed significantly higher survival percentages when compared with the negative control. Seed cryopreservation was performed successfully with a clear reduction in germination percentage in transgenic lines overexpressing high levels of SlAREB1. In conclusion, transgenic tomato lines overexpressing SlAREB1 were found to improve tolerance against different abiotic stresses associated with different in vitro preservation protocols.

Medium-Term Conservation of Achillea Fragrantissima Forssk Sch. Microshoots = الحفظ متوسط الأمد لسويقات نبات القيصوم البري Achillea Fragrantissima Forssk Sch.

The harvest of Achillea fragrantissima on a mass collection from their natural habitats in Jordan is causing a decline of total plant genetic resources. So the conservation of this important genotype is vital. Slow growth conservation was used in this study. It based on culturing of the plant material on elevated concentrations (0.0, 0.1, 0.2, 0.3, or 0.4 M) of different osmotic agents (sucrose, sorbitol, or mannitol) and elevated concentrations (0.0, 3.8, 7.6, or 11.4 µM) of abscisic acid. Conservation of A. fragrantissima microshoot on medium supplemented with 0.1 M of (sucrose, sorbitol, or mannitol), or 3.8 µM ABA under light at 25°C , was able to reduce the growth rate and maintain the plant for 12 weeks.

In Vitro Conservation and Cryopreservation of Medicinal and Aromatic Plants : A Review = حفظ النباتات الطبية و العطرية داخل الأنابيب و التجميد بالنيتروجين السائل : مراجعة

Medicinal and aromatic plants are those containing special chemical components that enable them to relief pain, release pleasant aromas, and improve food flavors. Meanwhile, medicinal and aromatic plants are under real jeopardy due to the uncontrolled collection of these plants as a result of being extensively used in herbal medicine and food industry. So conservation action for these natural resources is of high priority. Plants conserved under in situ conservation conditions are exposed to natural disasters, pests and pathogens in addition to the fluctuating government policies. Also ex situ conservation is very difficult to be applied as adequate samples have to be collected for the conservation of genetic diversity. In vitro conservation is offering a strong and a multi package of techniques that do so good when other conservation methods are not feasible. Slow growth conservation is a very simple in vitro technique based on reducing the growth rates of the tissue cultured plant for short or mid- term storage and yet increasing the intervals between subcultures. In this conservation type, several techniques are used separately or in combinations to slow down the growth rate of the stored explants, such as, addition of elevated levels of osmotic agents or ABA in addition to storage under minimal growth conditions such as low temperature and dark incubation. Cryopreservation is another conservation technique that is usually described as the most reliable tool for long-term storage of plant germplasm and reported to be advantageous over most other conservation methods in terms of simplicity, applicability to a wide range of genotypes and ability to maintain the genetic stability of plant material. Thanks to the use of cryopreservation techniques, such as, encapsulation-dehydration, vitrification, encapsulation-vitrification, and droplet-vitrification, many medicinal plant species are conserved indefinitely for the next generations.

In Vitro Propagation and Acclimatization of Achillea Fragrantissima Frossk Sch. Bip = الإكثار الدقيق و الأقلمة لنبات القيصوم ( Achillea Fragrantissima Frossk Sch. Bip )

A. fragrantissima is one of the most Achillia species found in Jordan, it is known by local communities as Qaisoum or Qisum. Most effective method found to increase and conserve herbal plant is in vitro propagation. It is considered to be easier and more rapid for herbaceous plants than woody species. In the current study, in vitro propagation of A. fragrantissima rooting and acclimatization was studied. Complete germination of seeds (100%) was obtained in vitro on water-agar medium and developed into plantlets with greater hypocotyl and root length, and full cotyledonary leaves. Multiplication of mother stock was established on MS (Murashige and Skoog) media supplemented with 1mg/L GA3 after 4 weeks of culture. Proliferation was experimented with different levels of cytokinin (0.0, 0.4, 0.6, 0.8, 1.2 or 1.6 mg/L) of BA, kinetin or 2ip. Maximum proliferation of Achillea fragrantissima (8 shoot/explant) was obtained when MS medium supplemented with 1.2 mg/L kinetin. Rooting was experimented at different levels of auxins (0.0, 0.4, 0.6, 0.8, 1.2, or 1.6 mg/L) of NAA, IAA or IBA. Highest root number (9.80) and length (1.80 cm) was obtained at 0.4 mg/L NAA, while IAA failed to promote root induction. Rooted plantlets were successfully acclimatized with 100% survival.