scholarly journals Optimization of regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni): a commercially important natural sweetener plant

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pooja Taak ◽  
Siddharth Tiwari ◽  
Bhupendra Koul
Keyword(s):  
Transgenic Plants ◽  
Blot Hybridization ◽  
Stevia Rebaudiana ◽  
Southern Blot Hybridization ◽  
Nodal Explants ◽  
Naphthalene Acetic Acid ◽  
Steviol Glycosides ◽  
Root Induction ◽  
Commercially Important

Abstract Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2–25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l−1) + naphthalene acetic acid (0.5 mg l−1), gibberellic acid (1.0 mg l−1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l−1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

scholarly journals High Frequency Plant Regeneration System of Aerides odorata Lour. Through Foliar and Shoot Tip Culture

2013 ◽  
Vol 41 (1) ◽  
pp. 169 ◽  
Author(s):  
Huidrom Sunitibala DEVI ◽  
Sanglakpam Irabati DEVI ◽  
Thingbaijam Dikash SINGH
Keyword(s):  
Shoot Tip ◽  
Naphthalene Acetic Acid ◽  
Root Induction ◽  
Regeneration System ◽  
Direct Shoot Regeneration ◽  
Leaf Base ◽  
Clonal Multiplication ◽  
Commercially Important ◽  
Shoot Tip Explants

An efficient protocol for rapid clonal propagation from different explants of Aerides odorata Lour.- an endemic orchid of Manipur has been established. Leaf base explants showed significant response in ½ strength Murashige and Skoog medium supplemented with thidiazuron (TDZ) and 6-benzylaminopurine (BAP). Callus were initiated only from leaf base explants after 60 days of culture while other parts of leaves failed to response in all the treatments. Medium supplemented with 1.0 mg/L TDZ produced protocorm like bodies (PLBs) at the leaf base. Shoot tip explants of A. odorata showed different morphogenetic responses in different phytohormone treatments. Calli were initiated only in the medium containing α-naphthalene acetic acid (NAA). Highest calli frequency was observed in the medium containing 2 mg/L (NAA) (85.71±0.21) which indicates the importance of exogenous auxin in embryonic callus proliferation. Direct shoot regeneration on the other hand was observed in all the treatments. Highest number of shoot was obtained in higher concentration of NAA (2 mg/L) and BAP (4 mg/L) (4.80±0.18), showing combined effect of BAP and NAA, which may be due to the synergistic effect of cytokinin and auxin. Among the different rooting phytohormones, addition of NAA (0.5 mg/L) in ½ MS medium shows highest frequency of root induction. More than 95% in vitro plants survived during acclimatization under ex vitro conditions. This phytohormones and explants based micropropagation system can open up the route for in vitro clonal multiplication of this commercially important Aerides species.

scholarly journals Release Kinetic Studies of Stevia rebaudiana Extract Capsules from Sodium Alginate and Inulin by Ionotropic Gelation

2018 ◽  
Vol 2018 ◽  
pp. 1-8
Author(s):  
Juan Pablo Quintal Martínez ◽  
Jorge Carlos Ruiz Ruiz ◽  
Maira Rubí Segura Campos
Keyword(s):  
Sodium Alginate ◽  
Release Kinetics ◽  
Stevia Rebaudiana ◽  
Kinetic Studies ◽  
Release Kinetic ◽  
Steviol Glycosides ◽  
Ionotropic Gelation ◽  
Diffusion Controlled ◽  
Release Analysis

This study was oriented towards encapsulation of S. rebaudiana extract and the study of its release kinetics. The desired encapsulation was achieved by the ionotropic gelation method using sodium alginate and inulin of polymeric constituents. Characterization of the capsules was performed by micrometric properties, encapsulation efficiency, in vitro extract release analysis, and biological activity of released extract. The in vitro release profiles from different capsules were applied on different kinetic models. The prepared capsules were found spherical in shape with diameters ranging from 2.07 to 2.63 mm, having the encapsulation efficiencies of 43.77% and 56.53% for phenolic compounds and steviol glycosides, respectively. The best-fit model with the highest correlation coefficient was observed in the Ritger–Peppas model, indicating diffusion controlled principle. The release exponent n value obtained from the Korsmeyer–Peppas model varied between 0.2273 and 1.1719, confirming that the mechanism of S. rebaudiana extract bioactive compounds release was diffusion controlled.

scholarly journals In vitro Shoot Cultures of Tupistra albiflora K. Larsen

2018 ◽  
Vol 17 (5) ◽  
pp. 405-411
Author(s):  
Jiraporn PALEE
Keyword(s):  
Agar Medium ◽  
Multiple Shoots ◽  
Shoot Formation ◽  
Multiple Shoot ◽  
Naphthalene Acetic Acid ◽  
Root Induction ◽  
Shoot Cultures ◽  
Ms Medium ◽  
In Vitro Shoots

To evaluate an efficient protocol for the micropropagation of Tupistra albiflora K. Larsen, the effects of N6-benzylaminopurine (BA) and naphthalene acetic acid (NAA) concentrations on multiple shoot and root induction were examined. In vitro shoots were used as the explant materials which were cultured on Murashige and Skoog (MS) agar medium supplemented with 0, 1, 2, 3 and 4 mg/L BA for 4 weeks to induce multiple shoots. It was found that the MS medium containing 3 mg/L BA induced 100 % shoot formation with the highest number of 3.2 shoots per explant (2.4-fold significantly higher than the control). For root induction, in vitro shoots were cultured on MS agar medium supplemented with 0, 1, 2, 3 and 4 mg/L NAA for 8 weeks. The results showed that the MS medium containing 1 mg/L NAA induced 100 % root formation with the highest number of 6.6 roots per explant (1.8-fold significantly higher than the control).

scholarly journals Factors affecting organogenesis of Stevia rebaudiana and in vitro accumulation of steviol glycosides

2020 ◽  
Vol 107 (2) ◽  
pp. 171-178
Author(s):  
Aušra Blinstrubienė ◽  
Natalija Burbulis ◽  
Neringa Juškevičiūtė ◽  
Rasa Žūkienė
Keyword(s):  
Stevia Rebaudiana ◽  
Steviol Glycosides ◽  
Factors Affecting

Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination

1983 ◽  
Vol 3 (11) ◽  
pp. 1943-1948
Author(s):  
L J Kelly ◽  
R Kelly ◽  
H L Ennis
Keyword(s):  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Developmental Regulation ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Molecular Weights ◽  
Multiple Copies

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.

In vitro mass propagation of Piper betle L

2019 ◽  
Vol 48 (3) ◽  
pp. 559-566
Author(s):  
Salim Khan ◽  
Barna Goswami ◽  
Shahina Akter ◽  
Mousona Islam ◽  
Afsana Huq Noon ◽  
...  
Keyword(s):  
Nodal Explants ◽  
Direct Regeneration ◽  
Leaf Segment ◽  
Piper Betle ◽  
Nodal Segment ◽  
Root Induction ◽  
Regeneration System ◽  
Petiole Explants ◽  
Best Response

An efficient in vitro regeneration system was developed for Piper betle L. through direct and indirect organogenesis from nodal segment, leaf segment and petiole explants. Highest direct regeneration was recorded when nodal explants were cultured on MS with 1.0 mg/l BAP and 1.0 mg/l Kn where 80% explants produced multiple shoots and the average number of shoots per explants were 3.20. Remarkable results on callus induction and shoot initiation were observed when the explants cultured on MS + 2.0 mg/l BAP + 0.5 mg/l Kn + 1.0 mg/l IAA. It was observed that nodal explants were showed best response on shoot/explants 13.2 ± 4.5 after 8 weeks of callus culture on MS medium with 0.5 mg/l BAP. The best response towards root induction was observed on half strength of MS with 0.25 mg/l IBA. The well rooted plants were successfully acclimatized and transferred to soil.

scholarly journals Phytohormones Influence on the In-Vitro Regeneration of Saccharum Officinarum L. from Apical Meristem Culture

2014 ◽  
Vol 5 (2) ◽  
pp. 85 ◽  
Author(s):  
Ejiroghene Felix Lawyer ◽  
Z. O. Jamaleddine ◽  
P. T. Lyam ◽  
I. T. Borokini ◽  
A. A. Adedeji ◽  
...  
Keyword(s):  
Plant Growth ◽  
Plant Growth Regulators ◽  
Growth Regulators ◽  
Apical Meristem ◽  
In Vitro Regeneration ◽  
Saccharum Officinarum ◽  
Naphthalene Acetic Acid ◽  
Root Induction ◽  
Indole Butyric Acid

Growth regulators especially auxins and cytokinins are critical for plant in-vitro regeneration. The effect of these plant growth regulators on in-vitro propagation of Saccharum officinarum L (Sugarcane) was investigated. In vitro response of two different varieties of sugarcane (NCS 005 and NCS 008) to Plant Growth Regulators was obtained in this study. Formation of buds was obtained on shoot apical meristem when cultured on MS (Murashige and Skoog) medium supplemented with 0.1mg/l BAP (6-Benzylaminopurine). After two weeks of initiation, regenerated meristem was inoculated into MS (Murashige and Skoog) fortified with different concentrations and combination of cytokinins. Shoot multiplication was optimal on 0.5mg/l BAP + 0.25 mg/l Kin(Kinetin) for NCS 005 variety while for NCS 008 variety, no significant (P≥0.05) difference was observed between 1.5mg/l BAP and 1.5mg/l BAP +0.5mg/l Kin. The best root induction for in vitro derived shoots was obtained on 1.0 mg/l NAA (Naphthalene acetic acid) and 2.0 mg/l IBA( Indole butyric acid) for both varieties of sugarcane within ten days of culture transfer. Successfully established plantlets showed excellent growth response when weaned under regulated green house conditions.

scholarly journals In vitro propagation of Citrullus lanatus Thumb. from nodal explants culture

1970 ◽  
Vol 8 (2) ◽  
pp. 203-206 ◽  
Author(s):  
MM Khatun ◽  
MS Hossain ◽  
MA Haque ◽  
M Khalekuzzaman
Keyword(s):  
Citrullus Lanatus ◽  
In Vitro Regeneration ◽  
Shoot Proliferation ◽  
Multiple Shoot ◽  
Nodal Explants ◽  
Root Induction ◽  
Ms Medium ◽  
Nodal Explant ◽  
Grown Plant

A standard protocol was established for rapid in vitro propagation of watermelon (Citrullus lanatus Thumb.) from nodal explants of field grown plant. Multiple shoot proliferation was achieved from nodal explants on MS medium supplemented with 1.0 mg/l BAP + 0.2 mg/l NAA within 30 days of inoculation. The elongation of shoots was obtained on the same medium. Highest percentage of root induction was achieved on MS medium supplement with 1.0 mg/l IBA within 25 days of culture. Well rooted plantlets were transferred to small pots and after proper acclimatization the plantlets were transplanted in the field condition, where 80% plantlets were survived and grew successfully. Keywords: In vitro regeneration; Nodal explant; Citrullus lanatus DOI: 10.3329/jbau.v8i2.7926 J. Bangladesh Agril. Univ. 8(2): 203-206, 2010  

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