Effect of cytokinin types, concentrations and their interactions on in vitro shoot regeneration of Chlorophytum borivilianum Sant. & Fernandez

Background: Chlorophytum borivilianum is a rare medicinal plant originally distributed throughout the forest of India. The tubers of C. borivilianum are used as an aphrodisiac and impotence supplement. The propagation of C. borivilianum is possible through seeds and tubers, but conventional methods may take several months. Hence in vitro technique of shoot regeneration could be an efficient alternative means of propagating the species. Latest study reported microtuberization of C. borivilianum but there is no sufficient study on a rapid method for shoot multiplication and elongation. Results: Young shoot buds of C. borivilianum were cultured on MS medium containing 6-benzylaminopurine (BAP) and Kinetin (Kn), both at 0, 8.88, 17.8 and 26.6 \u3bcM, either individually or in combinations. Proliferated shoots were subcultured on fresh medium of the same constituents on week 3 of culture for further shoot multiplication and elongation. BAP alone (8.88\u201326.6 \u3bcM) was significantly effective on shoot multiplication, while Kn alone (8.88\u201326.6 \u3bcM) was significantly effective on shoot elongation compared to the control containing MS basal medium without any plant growth regulator. However, combination of both cytokinins stimulated an interaction producing higher shoot number and shoot length compared to their individual application. Conclusions: The most suitable combination was 8.88 \u3bcM BAP + 8.88 \u3bcM Kn, reaching a mean shoot number of 10.83 and shoot length of 6.85 cm

In vitro mutagenesis using bio-beam irradiation on in vitro culture of Cavendish banana cultivar (Musa acuminata Colla) explants

Banana, Musa acuminata cv. Cavendish, was commonly propagated by use of suckers from selected field grown clumps. An in vitro technique has in recent years provided with rapid and efficient system propagation. However, high demand for planting material has caused significant shortages and subsequently attempts at improving the efficiency of in vitro propagation technique have been initiated. The current study investigated the effect of gamma irradiation using bio-beam on in vitro propagation of banana cv. Cavendish. The main objective was to assess the effect of mutation induction using gamma source on growth and development of plantlets while in culture. Tissue cultured banana plantlets were used as source of explant materials. The plantlet was trimmed of leaves and halved before culturing on Murashige and Skoog (MS) media. Then, the explants radiated with gamma source using Ion-Beam with selected dose [0 (control), 30 Gy, 60 Gy, 90 Gy, 120 Gy and 150 Gy] and transferred to fresh MS media with 5 mg/l of BAP. The analysis showed dosage of 30 Gy and 60 Gy produced significant survival rate and favourable effect on growth especially on plant height, shoot number, leaf production and root production. Thus, this study contributed to develop in vitro mutagenesis of M. acuminata cv. Cavendish through gamma rays coupled with in vitro system for future breeding of new varieties of banana

Effect of cytokinin types, concentrations and their interactions on in vitro shoot regeneration of Chlorophytum borivilianum Sant. & Fernandez

Background: Chlorophytum borivilianum is a rare medicinal plant originally distributed throughout the forest of India. The tubers of C. borivilianum are used as an aphrodisiac and impotence supplement. The propagation of C. borivilianum is possible through seeds and tubers, but conventional methods may take several months. Hence in vitro technique of shoot regeneration could be an efficient alternative means of propagating the species. Latest study reported microtuberization of C. borivilianum but there is no sufficient study on a rapid method for shoot multiplication and elongation. Results: Young shoot buds of C. borivilianum were cultured on MS medium containing 6-benzylaminopurine (BAP) and Kinetin (Kn), both at 0, 8.88, 17.8 and 26.6 μM, either individually or in combinations. Proliferated shoots were subcultured on fresh medium of the same constituents on week 3 of culture for further shoot multiplication and elongation. BAP alone (8.88–26.6 μM) was significantly effective on shoot multiplication, while Kn alone (8.88–26.6 μM) was significantly effective on shoot elongation compared to the control containing MS basal medium without any plant growth regulator. However, combination of both cytokinins stimulated an interaction producing higher shoot number and shoot length compared to their individual application. Conclusions: The most suitable combination was 8.88 μM BAP + 8.88 μM Kn, reaching a mean shoot number of 10.83 and shoot length of 6.85 cm

Female reproductive system of Amaranthus as the target for Agrobacterium-mediated transformation

Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy time for screening transformed progenies. Therefore, in this study, three constructs, p5b5 (14,289 bp), p5d9 (15,330 bp) and p5f7 (15,380 bp) in pDRB6b vector which has hygromycin as a selectable marker gene were introduced individually into Agrobacterium tumefaciens strain (AGL1). The cell suspension was applied to Amaranthus inflorescence by drop-by-drop technique and was left to produce seeds (T1). The T1 seeds were germinated and grown to produce seedlings under non-sterile condition. Hygromycin selection on seedling cotyledon leaves results in identification of 12 putative transformants, three from p5b5, four from p5d9 and five from p5f7. All positive putative transformants that were selected at the first stage through hygromycin spraying showed positive result in leaf disk hygromycin assay and in a construct specific polymerase chain reaction-based assay. A ~750 bp amplified hygromycin gene was further verified through sequencing. Our results suggest that Amaranthus inflorescences were able to be transformed and the transformed progenies could be verified through a combination of simple and rapid methods

In vitro propagation of Etlingera elatior (Jack) (torch ginger)

An efficient protocol for complete plant regeneration from suckers of Etlingera elatior (Jack) has been developed. The addition of N6-benzyl amino-purine (BAP) (0, 13.32, 22.20, 31.08, and 44.40 μM) to the culture medium comprising Murashige and Skoog (MS) basal salts, 3% sucrose, 0.4% Gelrite™ did not show any significant effects on percentage of shoot induction and mean number of shoots produced. Various concentrations of BAP (0, 13.32, 22.20, 31.08, and 44.40 μM), 6-furfurylaminopurine (kinetin) (0, 13.95, 23.25, 32.55, and 46.50 μM) and N6-(2-isopentenyl) adenine (2-iP) (0, 14.76, 24.6, 34.44, and 49.2 μM) were tested for shoot multiplication. BAP at all levels were found suitable for the multiplication of shoot. However, the low level of 13.32 μM BAP was chosen as the best concentration of BAP due to economic feasibility. The best root proliferation was observed on MS medium without plant growth regulator (PGR). Assessment of various potting media for acclimatization showed medium containing soil:sand:peat moss (1:1:1) produced high survival of plantlets, number of leaves produced per plant and the plant height. This protocol provides a basis for future studies on mutation induction, genetic engineering, and could be applied for large-scale multiplication of this valuable plant

An Efficient Agrobacterium-Mediated Transformation of Strawberry cv. Camarosa by a Dual Plasmid System

An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100%) of shoot formation and the highest mean number of shoots (24) produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86%) in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet