Highest Frequency Of Shoot Formation Research Articles

Photosynthetic Effects of Light-emitting Diode (LED) on in Vitro-derived Strawberry (Fragaria x Ananassa cv. Festival) Plants Under in Vitro Conditions

Purpose of this study was to investigate the effect of different photosynthetic photon flux density (PPFD) conditions using LED lamps on culture of shoot explants derived from in vitro shoots of Fragaria x ananassa cv. Festival. To examine the combined effect of 55% red LED, 15% far red LED, 10% blue LED, 15% green LED and 5% warm light LED light on in vitro growth of plantlets, fresh and dry plant biomass, plant height, leaf area, number of shoot, shoot length, number of root and percentage of rooting and various growth of micro-propagated plants were assessed under four light intensities (25, 50, 75 and 100 μmol m−2 s−1 PPFD; TRT2, TRT3, TRT4, TRT5, respectively). Un-rooted strawberry shoots were cultured in the “Culture Pack”-rockwool system with MS medium under CO2-enriched condition. The best response for regeneration of shoots and root induction was observed for shoot explants obtained on MS supplemented with BAP 1 mg/l and NAA 0.5 mg/l at TRT3 (50 μmol m−2 s−1 PPFD). Chlorophyll and net photosynthesis were optimal in plants grown under TRT4 (75 μmol m−2 s−1 PPFD). Stomatal resistance and Fv/Fm values were highest at low light irradiance (TRT2). The highest efficiency and high frequency of shoot formation occurred after 30 days. Elongation of shoot buds was obtained at TRT4 (75 μmol m−2 s−1 PPFD) and TRT5 (100 μmol m−2 s−1 PPFD) on the same MS medium. Regenerated shoots rooted best on the same medium of elongation. Irradiance at 75 μmol m−2 s−1 PPFD was suitable for the acclimatization of strawberry plants. We concluded that the present protocol can be efficiently used for mass propagation of the strawberry.

Influence of cold pretreatment on shoot regeneration from callus in date palm (Phoenix dactylifera L.) cv. ‘Barhee’

Mass propagation of date palm through indirect somatic embryogenesis or organogenesis has attracted the interest of commercial producers. But, this technique still faces some problems that hindered the production of date palm plantlets in vitro. Tissue browning is one of the serious problems that reduce callus growth and shoot regeneration. So the objective of the present study is to investigate the effect of cold pretreatment on callus growth, shoot regeneration, and polyphenol oxidase (PPO) activity during the callus culture. Results showed that a high survival rate of callus cultures (100%) were obtained when cultures were incubated in low temperature (cold treatment) for 45 and 75 days. On the other hand, total amount on phenolic compounds was also reduced to 0.47 and 0.53 mg GAE/g after same period of incubation (45 and 75 days respectively) at low temperature. In additional, our results showed that the highest frequency of shoot formation (66.67 and 73.34, %) and the highest shoot numbers (7.8 and 8.6 shoots/100 mg) were obtained from callus treated with low temperature for 45 and 75 days, respectively.

Effect of Genotypes and Sugar Type on Callus Formation and Plant Regeneration Derived from Mature Grains of Egyptian Rice (Oryza sativa L.)Varieties

The main objective of the present study was to determine the highest regenerable Egyptian rice cultivars and the best source of carbohydrates. Three medium protocols had been used to determine the in vitro culture response of mature dehusked grains in five Egyption rice cultivars. Factorial experiment in CRD design with ten replications was used to analyze the data. The obtained results showed that in vitro traits were highly significantly affected by genotypes and the interaction between medium protocols and genotypes. Whereas, medium protocols had different effects on the in vitro traits. The results also indicated that the shoot formation (%) and number of shoots per callus traits were highly significantly affected by genotypes, medium protocols and the interaction between medium protocols and genotypes. In conclusion, the cultivars, Sakha 103 and Sakha 104 could be successfully utilized in breeding programs for biotic and abiotic stress tolerance at the cellular level, because of their high frequency of shoot formation on the medium protocol C.

Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui

Ceropegias has acquired significant importance due to their medicinal properties, edible tubers, and its ornamental flowers. The aim of this study was to optimize direct shoot organogenesis (DSO), indirect shoot organogenesis (ISO) and plant regeneration of threatened medicinal plant Ceropegia santapaui, followed by analysis of genetic status and biochemical characterization of micropropagated plantlets. For optimization, cotyledonary nodes and cotyledons were used as source of explants in DSO and ISO respectively. The highest frequency of regeneration (88.0 %) for DSO with 8.1 ± 0.6 shoots per explant was obtained from cotyledonary nodes cultured on Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 2iP. The best response for callus induction and proliferation was achieved with 1.5 mg L−1 PR (picloram) in which 97.5 % of cultures produced an average of 913 ± 10.9 mg (fresh weight) of callus. The highest frequency of shoot formation (92.5 %) with an average of 19.7 ± 0.3 shoots in ISO was obtained when calli were transferred to MS medium supplemented with 2.5 mg L−1 BAP and 0.4 mg L−1 IBA. Regenerated shoots were best rooted in half-strength MS medium with 2.0 mg L−1 NAA. Plantlets successfully acclimatized were morphologically indistinguishable from the source plant. Micropropagated plantlets subjected to random amplified polymorphic DNA and inter simple sequence repeats (ISSR) marker based profiling reveled uniform banding pattern in DSO-derived plantlets which was similar to mother plant. ISSR fingerprints of ISO-derived plants showed low variation. Method of regeneration, plant part and solvent system significantly affected the levels of total phenolics, flavonoids and antioxidant capacity. Assay of antioxidant activity of different tissues revealed that significantly higher antioxidant activity was observed in ISO-derived tissues than DSO-derived and mother tissues. RP-HPLC analysis of micropropagated plantlets showed the presence of three major phenolic compounds which were similar to those detected in mother plant. Rapid multiplication rate, genetic stability and biochemical parameter ensures the efficacy of the protocol developed for the propagation of this threatened medicinal plant.

Micropropagation and root culture of Turkish endemic Astragalus chrysochlorus (Leguminosae)

Efficient micropropagation and root culture protocols were developed for the endemic Astragalus chrysochlorus Boiss. & Kotschy. A high frequency of shoot formation (100%) and maximum multiplication (13 shoots per hypocotyl explant) were achieved on Murashige and Skoog (MS) media supplemented with 0.5 mg/L trans-Zeatin riboside (ZR). By using single regenerated hypocotyl explants, rooting was achieved at a rate of 93% on MS medium containing 2% sucrose without growth regulators. High frequency callus initiation and growth were achieved when single hypocotyl explants were inoculated on MS medium supplemented with 0.5 mg/L 2,4-Dichlorophenoxyacetic acid. Plant regeneration through indirect organogenesis was achieved on MS medium supplemented with 0.5 mg/L ZR. Root cultures were successfully established in liquid MS medium containing 0.5 mg/L \alpha-Naphthaleneacetic acid. The optimised in vitro propagation, callus culture, and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies on Astragalus L. species.

In vitro regeneration of Solanum nigrum with enhanced solasodine production

High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Skoog (MS) medium without any callusing stage. Shoot forming ability was more pronounced on leaves positioned dorsally. For shoot induction, 2.0 mg dm-3 benzylaminopurine and 1.5 mg dm-3 kinetin were observed to be the most effective plant growth regulators (PGRs). The present paper also describes first successful induction of in vitro flowering in S. nigrum. The leaf derived shoots were excised and treated with various root promoting PGRs and 0.25 mg dm-3 indole-3-butyric acid produced maximum number of roots (15.2 per plant). Plants were later transplanted in field with 100 % survival. Solasodine content was higher in in vitro raised shoots and leaf derived callus, compared to ex vitro grown shoots.

Changes of antioxidant enzyme activities and isoenzyme profiles duringin vitroshoot formation in saffron (Crocus sativusL.)

Among the different concentrations of Thidiazuron (TDZ) and between the two media Gamborg (B5) and Murashige and Skoog (MS), the highest frequency of shoot formation could be seen in the MS medium with TDZ concentration of 4.54 microM. Among the different concentrations of Naphtalene acetic acid (NAA) and Benzyl adenine (BA) in the two aforementioned media, the maximum proliferation and rooting of saffron shoots were obtained in a B5 medium containing 2.22 microM NAA and 2.68 microM BA. Peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), esterase (EST) and polyphenoloxidase (PPO) measurements proved that all the enzymes had a similar pattern of changes, according to which their concentrations increased in the first stages of development and then decreased. The same pattern was observed for polyphenoloxidase in a B5 medium while in the MS medium a reverse pattern was observed. The enzyme concentration decreased and then increased during shoot formation. The results show the principal role of antioxidant enzymes in the complicated process of organogenesis.

할미 꽃 (Pulsatilla koreana NAKAI) 식물 절편체로부터 부정아 유도에 미치는 cytokinin의 영향 및 식물체 재 분화

Leaf and petiole explants of Pulsatilla koreana NAKAI were cultured on MS medium supplemented with various concentrations of zeatin, kinetin or BAP combined with 0.05 mg/L IAA. After 6 weeks of culture, effects of cytokinin on adventitious shoot formation from explants were investigated. The highest frequency of shoot formation was obtained when petiole explants were cultured on medium with 0.5 mg/L zeatin and 0.05 mg/L IAA. Regenerated shoot were transferred on to root induction medium. The best root formation was observed at 1/2 MS medium with 1.5 mg/L NAA. Rooted plantlets were transplanted to a mixture of perlit and soil (1:3), where they were successfully acclimatized.

Transgenic spinach plants produced by Agrobacterium-mediated method based on the low temperature-dependent high plant regeneration ability of leaf explants

Agrobacterium strain EHA101 harboring plasmid pIG121-Hm containing the genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase (nptII) was co-cultivated with leaf explants from in vitro grown spinach plants. Hygromycin-resistant calli were obtained 1 month after selection of transformed cells on 2.5 g l−1 gellan gum-solidified MS medium containing 30 g l−1 sucrose, 0.5 mg l−1 2,4-D, 2 mg l−1 kinetin, 20 mg l−1 hygromycin and 20 mg l−1 meropenem trihydrate. Regeneration of adventitious shoots was affected by culture temperature, and the highest frequency of shoot formation was 40% when the calli were cultured at 14°C during regeneration process on 2.5 g l−1 gellan gum-solidified MS medium containing 30 g l−1 sucrose, 0.01 mg l−1 2,4-D, 1 mg l−1 kinetin, 1 mg l−1 gibberellic acid (GA3), 20 mg l−1 hygromycin and 10 mg l−1 meropenem trihydrate. Stable expression of gus gene was indicated by histochemical GUS assay in the leaves and roots of putative transgenic plants. Southern blot analysis of genomic DNA isolated from T0 plants confirmed the successful integration of T-DNA into the plant genome. Segregation of the gus gene in transgenic T1 progeny was confirmed by PCR analysis. These results show that Agrobacterium-mediated transformation combined with plant regeneration at a low temperature, 14°C, can be efficiently used for producing transgenic spinach plants with useful genes.

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized.

PLANT REGENERATION FROM LEAF EXPLANTS AND EFFICIENT AGROBACTERIUM-MEDIATED TRANSFORMATION SYSTEM OF CHRYSANTHEMUM (DENDRANTHEMA GRANDIFLORUM)

Leaf explants of chrysanthemum (Dendranthema grandiflorum) were cultured on MS medium supplemented with various concentrations of NAA, IAA, 2,4-D and BA. The highest frequency of shoot formation was observed on MS medium supplemented with 1.0 mg L -1 BA, 0.3 mg L -1 2,4-D in Puma and with 2.0 mg L -1 NAA, 0.5 mg L -1 BA in Subangryeok. Suitable concentrations of kanamycin to select transformants were 20 mg L -1 in Puma, and 50 mg L -1 in Subangryeok. In Puma, the calli formation was most efficient in the medium containing kanamycin from leaf discs co-cultivated with A. tumefaciens inoculation at 1:50 dilution ratio for 5 min. PCR detection of the npt II gene gave a 700 bp band in two transformants.

Plant regeneration from callus cultures of Maclura tinctoria, an endangered woody species

Some native species produce seeds with a low frequency of germination accompanied with a period of dormancy. These features make it difficult to produce new phenotypes through sexual propagation. Maclura tinctoria has been considered an endangered species due to extensive use of its wood and low frequency of seed germination. The objective of the present study is to establish an in vitro propagation system for this species. Organogenic friable callus formation from nodal segments has been obtained using woody plant medium (WPM) supplemented with 10.74 μM 1-naphthaleneacetic acid (NAA)+4.43 μM 6-benzylaminopurine (BA). Results indicate that the highest frequency of shoot formation is observed when WPM supplemented with 4.03 μM NAA+4.43 BA is used. For root formation, the use of WPM medium (pH adjusted to 7.0) supplemented with 23.62 μM indole-3-butyric acid (IBA) and 4.7gl−1 activated charcoal is recommended. For acelimatization, subjecting rooted plantlets to 70%, 50%, and 30% mesh screen, each successively for a period of 7 d, has resulted in 97% plantlet survival.

Transgene expression in broccoli (Brassica oleracea var. italica) clones propagated in vitro via leaf explants.

We have developed an efficient protocol for the in vitro propagation of transgenic broccoli plants using leaf explants as starting material. A high frequency of shoot formation from leaf explants was obtained on Murashige and Skoog medium containing benzyladenine (BA, 5 mg/l) and naphthaleneacetic acid (0.5 mg/l). Frequent subcultures of existing shoots and shoot clusters to medium containing only BA (2 mg/l) promoted rapid shoot multiplication. The use of a 1:1 mixture of Agargel and Gelrite in the rooting medium increased the number of healthy roots per rooted plant. Applying this protocol, we obtained thousands of clonal rooted plantlets within 6 months from a transgenic broccoli plant carrying the cry1Ac and cry1C genes from Bacillus thuringiensis associated with kanamycin and hygromycin selectable markers, respectively. Thirty randomly selected clones that had been propagated for 1 year on medium containing kanamycin (50 mg/l) all showed resistance to both kanamycin and hygromycin. Genomic DNA and total soluble proteins were isolated from 16 of these clones. Polymerase chain reaction analysis indicated that the cry1Ac and cry1C genes were both maintained. ELISA assays showed that all of the clones produced a high level of Cry1Ac protein similar to the original transgenic plant; however, most clones had significantly lower levels of Cry1C protein than the original plant. This variation indicates that it is important to evaluate transgene expression in transgenic clones propagated long-term in vitro. In vitro propagation starting from leaf explants was also successful with other transgenic and non-transgenic Brassica oleracea materials, including broccoli, cauliflower, and collard.

Thidiazuron Induced Multiple Shoot Formation in Chickpea (Cicer arietinum L)

Thidiazuron (TDZ), a substituted urea with cytokinin-like activity stimulated shoot proliferation in chickpea (Cicer arietinum L). Embryonic axis with half portion of both cotyledons was more responsive explant, producing ∼22 shootslexplant at 1.0 µM TDZ concentration but higher concentration of TDZ (5.0 µM) reduced both the shoot proliferation and growth. An incubation time of 12 h at 1.0 µM TDZ was sufficient for induction of multiple shoot formation.TD2 induced high frequency of shoot formation as compared to BAP and also minor salts of MS medium played an important role in increasing the number of shoots. Roots could be induced in these shoots in MS medium supplemented with 0.5 µM IBA.

Regeneration of shoot-buds on hypocotyl of Linum seedlings: a stress-related response

Transient stress of different types effective in induction of shoot-buds on hypocotyl of seedlings are heat-, salt- and mineral-stress, ABA- and proline-treatment and a simple decapitation. Of these decapitation is known and the rest have been worked out for the first time. Heat- and salt-stress were dose dependent, ABA-treatment was effective, in induction of shoot-buds, when it was combined with a prior mineral-stress. In addition to their individual effect in promoting regeneration, there was synergism between different types of stress. Highest frequency of shoot formation occurred when mineral-stress was combined with decapitation.

In vitro studies on Pea (Pisum sativum L.): I. Callus formation, regeneration and rooting

Abstract Callus production, shoot formation via organogenesis and rooting of the regenerated shoots are reported in an Egyptian variety of Pisum sativum L. Calli were initiated from hypocotyl, leaf, root and mature embryo explants when cultured on MS medium containing B5 vitamins and supplemented with 2 mg/l 2,4-D+1 mg/l kin. Among the different types of explants, hypocotyl showed best potential for callus proliferation. Hypocotyl, leaf and immature cotyledon explants were used for shoot organogenesis. The best results of shoot formation were achieved when hypocotyl explants were cultured on MS-medium supplemented with 2 mg/l BA+1 mg/l NAA. However, immature cotyledon explants showed the highest frequency of shoot formation with 1 mg/l BA. Data of in vitro rooting showed that maximum root frequency occurred on culture medium containing half strength of MS salts, 40 g/l sucrose and 2 mg/l NAA.

Adventitious Shoot Regeneration in Sainfoin (Onobrychis viciifolia Scop.)

Cotyledon and hypocotyl explants from in vitro grown seedlings of sainfoin were cultured on a range of media to develop an adventitious shoot regeneration system suitable for transformation. In general, callus formation was not influenced by the explants used, however, hypocotyls appeared to have a better regeneration capacity than cotyledons. The highest frequency of shoot formation was achieved from hypocotyl segments cultured onto the Murashige and Skoog (MS) medium containing 0.5 mg/l 6-benzylaminopurine (BAP) and 0.2 mg/l a-naphthaleneacetic acid (NAA). Regenerated shoots rooted in half strength MS supplemented with either 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA.

Plant Regeneration and Thiophene Production in Hairy Root Cultures of Rudbeckia hirta L. Used as an Antagonistic Plant to Nematodes.

In Rudbeckia hirta L., an antagonistic plant to nematodes, hairy roots were induced by infection with a wild strain (A-5) of Agrobacterium rhizogenes. Hairy roots cultured in half-strength MS agar medium without phytohormones showed vigorous growth and extensive lateral branching. Mikimopine (opine) was detected in the extract of hairy root by paper electrophoresis. Adventitious shoots were induced on the surface of hairy roots after 30 to 50 days of transfer to half-strength MS agar medium supplemented with BAP at 0.5-10.0 mg/l. The highest frequency of shoot formation was obtained at 0.5 or 1.0 mg/l BAP in combination with 0.1mg/l NAA. Plants regenerated from hairy roots showed morphological alterations such as wrinkled leaves, small size of flowers and abundant lateral branching of roots. A nematocidal compound, α-terthienyl, was detected in the extract from lateral roots of the regenerant.

Clonal propagation of mature elite trees of Commiphora wightii

Elite trees of Commiphora wightii (Arnott) Bhandari were selected from the wild on the basis of their content of guggul, an oleoresin. The selected tree was micropropagated through forced axillary branching on Murashige and Skoog's (MS) medium supplemented with benzyladenine (BA) and kinetin. Highest frequency of shoot formation was achieved on MS medium supplemented with 17.8 μM BA, 18.6 μM kinetin, 100 mg l-1 glutamine, 10 mg l-1 thiamine HCL and 0.3% activated charcoal. Seasonal changes affected the shoot proliferating potential of the initial explants in vitro. Transfer of shoots to a medium containing a lower concentration of BA (1.8 μM) and kinetin (1.9 μM) before rooting markedly stimulated shoot elongation. Shoots could be rooted by treating them with both indoleacetic acid and indolebutryic acid for 24 h in darkness and transferring them to a low-salt basal medium with activated charcoal. After rooting, transfer to a half-strength White's (modified) medium was necessary for further development of the plantlet. Regenerated plantlets were successfully established in soil.