Micropropagation and In Vitro Inflorescence of Pentas lanceolata

In this study, different concentrations of 6-benzyladenine (BA) on in vitro shoot and inflorescence inductions of P. lanceolata were investigated. The in vivo and in vitro floral characteristics of this plant were also compared. Nodal explants of P. lanceolata were cultured vertically with the cut ends inserted into semi-solid Murashige and Skoog (MS) medium supplemented with 0, 0.5, 1, 2, 4, and 8 mg L–1 BA. The results showed that the explants formed the highest numbers of shoots even when cultured in MS basal medium without any addition of BA, while the shoots formed in the explants cultured in MS medium supplemented with 1 mg L–1 BA were the longest. No inflorescence was found in the shoots cultured in MS medium supplemented with 8 mg L–1 BA, while the highest percentage of inflorescence induction was found in the shoots cultured in the medium supplemented with 0.5 mg L–1 BA. The apperances of in vivo and in vitro flowers of P. lanceolata were the same in many aspects except that the number of flower/inflorescence formed was different. In addition, water accumulation was observed only inside the in vitro flowers. Water deposit in the long tubular structure of P. lanceolata flower could cause anther injury, suggesting that flowers developed in vitro may not always produce pollen.

Sterilization protocols and the effect of plant growth regulators on callus induction and secondary metabolites production in in vitro cultures Melia azedarach L.

Melia azedarach L. is a valuable source of antioxidants and secondary metabolites. This study is a first extensive report about the effect of different serialization protocols and plant growth regulators (PGRs) on explant disinfection efficiency, callus induction and secondary metabolites production and accumulation in callus cultures of M. azedarach L. In this regard, the effect of plant growth regulators on callus induction and secondary metabolites production were examined. In addition, different sterilization agents were evaluated for disinfection of chinaberry leaf explants. The results showed that the lowest percentage of explant contamination and browning with the highest percentage of callus induction and callus growth obtained with explants pretreated with benomyl (2 g/L) for 2 h and sterilized with 7% H2O2 for 10 min and NaOCl 2% (without pH adjustment) for 12 min. Although adjusting the pH of NaOCl to pH = 7 and 10 significantly reduced the microbial contamination and increased the percentage of contamination-free cultures of M. azedarach L., adversely influenced the explant viability and callus induction and growth. The highest percentage of callus induction obtained on the MS medium containing 3 mg/L NAA/2,4-D and 1 or 3 mg/L Kin/BAP, and the highest callus yield (1804.833 mg/explant) belonged to the MS medium supplemented with 5 mg/L 2,4-D and 5 mg/L Kin. The callus cultures grown on the MS medium supplemented with 3 mg/L NAA and 1 mg/L Kin produced the highest amount of Quercetin (2.06 mg/g fresh weight), Rutin (5.56 mg/g fresh weight) and Kaempferol (1.84 mg/g fresh weight).

Development of an Efficient In vitro Regeneration Protocol for Chrysanthemum (Chrysanthemum morifolium Ramat)

An efficient and rapid in vitro regeneration protocol was developed for chrysanthemum (Chrysanthemum morifolium Ramat) using two local varieties of Bangladesh namely, BARI Chrysanthemum-2 (BARI Chry-2) and local yellow (Y). MS medium supplemented with nine different concentrations and combinations of BAP and IAA was employed to optimize regeneration protocol using young in vitro derived leaf explants. Direct organogenesis was observed from the leaf explants on MS medium supplemented with 0.5 mg/l BAP and 2.0 mg/l IAA (T6) for both the varieties. This treatment (T6) induced shoot buds directly on the adaxial surface of the leaf providing the highest regeneration percentage (90% for BARI Chry-2 and 94.73% for Y), the highest number of shoot/explant (7.6 for BARI Chry-2 and 8.6 for Y) and maximum length of the shoot after six weeks (3 cm for BARI Chry-2 and 2.9 cm for Y) of culture. Explants with initially regenerated shoots were subculture on hormone free MS medium for shoot elongation after 4 weeks of their inoculation. During elongation of shoots, 90-95% of the regenerated shoots produced roots spontaneously in hormone free MS medium within 7-8 weeks of their inoculation. Rooted plantlets were transplanted to the field following hardening where 100% plantlets were survived and produced flower without any variation. Plant Tissue Cult. & Biotech. 31(2): 161-171, 2021 (December)

In Vitro Regeneration of Tea (Camellia sinensis (L). O. Kuntze) By Somatic Embryogenesis from Immature Cotyledon Tissues

Tea (Camellia sinensis) is the world's most popular beverage plant, as well as an important plantation crop with high commercial value. It has been maintained for centuries through conventional vegetative propagation. Tea clonal propagation in vitro has the advantage of producing a large number of elite plants. If an efficient in vitro regeneration technology is available, this technique could be exploited for selection of tea plants for desired trait. The selected plants could be later on multiplied through in vitro or ex vitro techniques. The study aimed to induced somatic embryogenesis from immature embryo explants to genetic variaton. Different concentrations of phenylboronic acid with benzyladenine and phenylboronic acid with kinetin were tested in MS medium with 30 g/L sucrose and 8 g/L agar. MS medium without any plant growth regulators was used as control group. Considering the embryo survival rate, 1.5 mg/ L-1 phenylboronic acid + 1 mg/ L-1 kinetin produced highest result as 87.3% while lowest was in control group as 36.7%. The highest plant regeneration rate was found in 1,5 mg/ L-1 phenylboronic acid + 1 mg/ L-1 kinetin and 1.5 mg/ L-1 phenylboronic acid + 1 mg/ L-1 benzyladenine medium respectively as 58.3% and 55.6%. Kinetin treatment with increasing phenylboronic acid concentrations gave the best results in terms of somatic embryo survival rate. Also, kinetin treatment produced better results when compared to benzyladenine concentrations.

Efficient Plant Regeneration via Indirect Organogenesis in Carnation (Dianthus Caryophyllus Semperflorens flore pleno) Cultivars

ABSTRACT Callus induction and plant regeneration are important steps of in vitro plant breeding of ornamental plants. In this study, the effects of different combinations of plant growth regulators (PGRs), promoters, and minerals on callus induction and plant regeneration in different carnation cultivars were studied in a completely randomized design with three replications. For callus induction, 16 different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and casein hydrolysate (CH) were studied using in vitro leaf explants. The Murashige and Skoog (MS) medium supplemented with 0.2 mg·dm-3 of 2,4-D and 200 mg·dm-3 of CH showed the highest frequency of callus induction. Among the cultivars, ‘Noblesse’ showed the highest rate of callus induction (91.67%). Regarding regeneration, BA, NAA, silver nitrate (AgNO3), and adenine hemisulfate (As) were used in ten different combinations. The ‘Cameron’, ‘Tabasco’, and ‘Noblesse’ cultivars with 95.24% regeneration percentage showed the highest rate of plant regeneration. Generally, in most cultivars, the highest regeneration rate and shoot number per explant were found in the MS medium supplemented with 3 mg·dm-3 of BA, 0.6 mg·dm-3 of NAA, 5 mg·dm-3 of AgNO3, and 40 mg·dm-3 of As. According to the results, the highest regeneration frequency was obtained when 40 mg·dm-3 of As was added to the medium. Finally, the flow cytometry analysis indicated that there were no significant differences between in vitro regenerated and control plants in terms of DNA ratios.

Effect of activated charcoal and ascorbic acid on in vitro morphogenesis and o-dihydroxyphenols content in Paphiopedilum insigne

Phenolic compounds limit micropropagation of many orchids in vitro. The aim of the study was to estimate the effect of activated charcoal (AC);1, 2 or 4 g/L) or ascorbic acid (AA; 10, 20 or 30 mg/L) added to the half strength MS medium on the growth and o-dihydroxyphenols content in Paphiopedilum insigne in vitro. A positive effect of AC on the shoot and root formation has been found. The highest multiplication rate (5.6 shoots/explant) and rooting frequency were obtained on medium containing 2 g/L of AC. However, AC reduced the leaf number as compared to the control. The lowest content of o-dihydroxyphenols was marked in Paphiopedilum insigne leaves when the shoots were grown on medium with 10 mg/L AA, followed by AC at 1 or 2 g/L.

Micropropagation of Feverfew (Tanacetum parthenium) and Quantification of Parthenolide Content in Its Micropropagated and Conventionally Grown Plants

Feverfew (Tanacetum parthenium) is a well-known multi-functional plant with anti-inflammatory, cardiotonic, antiangiogenic, and anticancer effects. The therapeutic value of this plant is due to its phytochemical constitutes, especially parthenolide. Tissue culture techniques have been applied to improve the bioactive components of many herbal plants. Hence, this study, was carried out to establish a protocol for micropropagation of the feverfew plant and to quantify parthenolide content in its micropropagated and conventionally grown plants. To establish an aseptic culture, different concentrations of sodium hypochlorite (NaOCl) were investigated for seed surface sterilization. Besides, the effects of plant growth regulators (PGRs) on the callus induction, shoot organogenesis from callus and in vitro rooting were evaluated. Additionally, the parthenolide yield of the micropropagated and conventionally grown plants was determined by using high-performance liquid chromatography (HPLC). The results showed that surface sterilization of feverfew seeds with 6% NaOCl for 15 min obtained 65.00 ± 2.69% aseptic seeds. Murashige and Skoog (MS) medium supplemented with 0.4 mg/L thidiazuron (TDZ) and 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) resulted in 86.00 ± 1.72% callus induction. The highest number of shoots (5.00 ± 0.15) per explant was obtained in the treatment of MS medium supplemented with 5 mg/L zeatin. MS medium fortified with 3 mg/L indole-3-butyric acid (IBA) produced the maximum number of roots per plantlet (8.90 ± 0.35). A total of 90% of the micropropagated plantlets survived when planted in perlite + peat moss (1:1 v/v); the micropropagated plantlets were successfully established in the ex vitro conditions. According to parthenolide analysis, its level was significantly higher in the micropropagated plants than conventionally grown plants. Among different solvents, ethanolic extraction obtained the highest parthenolide content of the feverfew plant. Hence, it can be concluded that micropropagation of feverfew could be applied to produce disease-free planting materials and to improve the parthenolide content of the feverfew plant.

BA and TDZ in the Morphogenesis in Vitro of Passiflora Setacea

Passiflora setacea DC (Passifloraceae) is considered an important species in the genetic breeding of passion fruit. However, its use is limited due to low seed germination. This paper aimed to study the effect of cytokinins 6-benzyladenine (BA) and thidiazuron (TDZ) on the in vitro morphogenesis of P. setacea using three explants: hypocotyl, nodal segment, and root segment. The explants were induced to morphogenesis in MS medium modified and with different concentrations of BA and TDZ. After 55 days, the percentage of calluses and shoots were evaluated, and anatomical characterization was performed. The three explants used in the in vitro morphogenesis of P. setacea showed callus and shoots formation, but in greater numbers in the nodal segments treated with BA. TDZ isolated affected equal to or less than BA on callus and shoots formation for the three explants. Direct and indirect organogenesis was observed in the three types of explants. From the results obtained for plant regeneration via in vitro morphogenesis of P. setacea, it is recommended to use a nodal segment in MSM medium and supplemented with 2.22 μmol L-1 of BA.

SLOW GROWTH IN VITRO CULTURE FOR CONSERVATION OF HANCORNIA SPECIOSA GOMES

Hancornia speciosa Gomes is a fruit species endemic to the Cerrado and coastal plains of Northeast of Brazil, with great economic, nutritional, ecological, and medicinal potential. This study aimed to evaluate the effect of sorbitol and sucrose as osmotic regulators on the in vitro growth of mangabeira, aiming at conservation by slow growth. The explants were obtained from in vitro germinated seedlings and inoculated in MS medium supplemented with sucrose (15 and 30 g L-1) and sorbitol (0, 5, 10 and 15 g L-1). The experimental design was completely randomized with 20 repetitions in a 4 x 2 factorial arrangement (sorbitol x sucrose concentrations). The evaluations were performed at 30, 60 90 and 120 days of incubation. The analyzed variables were number of nodes/budding, number of leaves, leaf abscission, leaf color and survival of explants. The data were statistically analyzed by generalized linear model analysis. The results indicated a significant difference between the osmotic regulators and the culture time for all variables. Sorbitol showed a more pronounced growth-reducing effect than sucrose. The use of 30 g L-1 sucrose combined with 10 or 20 g L-1 sorbitol reduced the growth in a critical way, making it clear that the water stress caused was not tolerated by the plants, negatively interfering in its development. Treatment with 15 g L-1 sucrose combined with 5 g L-1 sorbitol promoted the best result, allowing the conservation of plants for 120 days.

In vitro propagation from nodal segments of Lippia origanoides (chemotype A)

ABSTRACT: This research described an efficient micropropagation protocol for Lippia origanoides (Verbenaceae). Sterile seeds were used to obtain germinated seedlings in Murashige and Skoog medium (MS) supplemented with sucrose and agar. The nodal segments obtained from seedlings were grown on MS medium supplemented with different concentrations of gibberellic acid (GA), benzylaminopurine (BAP) and 1-naphthalenacetic acid (NAA) with BAP. The callus induction, shoots length, shoots number and root length, were analyzed. The treatments showed high percentage of callus formation at 0.5 to 1.5 mg L-1 of BAP alone or in combination with NAA (0.1 mg L-1). The highest value of shoot number per nodal segments was obtained at 1.5 mg L-1 of BAP (4.3 ± 0.8). The obtained plantlets were better rooted in vitro in the absence of plant growth regulators (PGRs) and they showed acclimatization rate of 90%. We reported a protocol for in vitro propagation and acclimatization of L. origanoides for A chemotypes from Colombia.