Highest Shoot Regeneration Frequency Research Articles

Indirect organogenesis for high frequency shoot regeneration of two cultivars of Sansevieria trifasciata Prain differing in fiber production

Sansevieria trifasciata is used as an indoor plant, in traditional medicine and as a fiber source. Here we characterized fibers of two of varieties of S. trifasciata, Lorentii and Hahnii, and report a protocol for their propagation based on indirect shoot organogenesis. Structural and ribbon fibers were scattered within leaf parenchyma when viewed with confocal laser scanning microscopy. Chemical analysis of the fibers by mass spectrometry and high-performance chromatography revealed higher contents of cellulose and xylose in Lorentii than in Hahnii and significant differences for total lignin between both. A protocol for de novo shoot production was then developed using leaf explants. Time-course histological analyses showed that the first events of transdifferentiation were triggered preferentially in cells surrounding fibers and vascular bundles. Callogenesis and shoot performances were quantified for both varieties, and 2,4-D at 2 and 3 mg·L-1 yielded the best results for primary calli induction and fresh calli mass. The length, number, and mass of shoots produced did not differ significantly between the two cultivars. The fast morphogenic response of S. trifasciata to in vitro culture may be useful for mass propagation or other biotechnological purposes such as metabolite production.

Combination of 6-Benzylaminopurine and Thidiazuron Promotes Highly Efficient Shoot Regeneration from Cotyledonary Node of Mature Peanut (Arachis hypogaea L.) Cultivars

Efficient in vitro plantlet regeneration is an important step to successfully transform genes for the improvement of agronomic traits. A combination of 6-benzylaminopurine (BAP) and thidiazuron (TDZ) plant growth regulators was applied to evaluate shoot regeneration capacity whereas α-naphthalene acetic acid (NAA) combination with 6-benzylaminopurine (BAP), and 2, 4-dichlorophenoxyacetic acid (2, 4-D) with 6-benzylaminopurine were tested to optimize root induction for two peanut cultivars. The result showed combination (BAP with TDZ) was found to be effective in promoting shoot. The highest shoot regeneration frequency (93%) was obtained on a medium supplemented with 4 mg/L BAP and 0.5 mg/L TDZ while an average regeneration frequency (87%) was achieved in a medium containing combinations of 2 mg/L BAP with 1 mg/L TDZ. The shooting rate increased for both cultivars as the concentrations of BAP increased and TDZ decreased. The highest rooting rate (93%) was obtained on a medium supplemented with 3.5 mg/L NAA with 2.5 mg/L BAP for both cultivars. The rooting rate increased as the concentration of auxin to cytokinin ratio increased. The maximum rooting rate (83%) was obtained on MS medium supplemented with 0.3 mg/L 2, 4-D with 0.2 mg/L BAP for the cultivar N3. The result indicated that BAP with NAA was much better than BAP with 2, 4-D in rooting rate. Thus, the protocol developed was genotype independent and effective for peanut tissue culture.

Metal Akümülatör Bitki olan Brassica nigra L.’nin in vitro Kallus İndüksiyonu ve Sürgün Gelişimi

This study established an efficient in vitro callus formation and plant regeneration protocol for a prevıously reported Cu accumulator, Brassica nigra, black mustard collected from Diyarbakir (Station site). Node explants from 10-day old mature plants were used for callus formation and shoot regeneration. The highest callus formation frequency (100%) was observed on Murasige Skoog (MS) medium containing 0.1 mg/L Benzylaminopurine (BAP) + 0.5 mg/L Naphthylacetic acid (NAA) (MS 2), 0.6 mg/L BAP + 0.2 mg/L NAA (MS 7), the highest shoot regeneration frequency (100%) was achieved on MS medium containing 0.6 mg/L BAP + 0.05 mg/L Indole butyric acid (IBA) (MS 8), 0.2 mg/L IBA + 0.2 mg/L NAA (MS 10) and the highest number of shoots per explant (3,25) was obtained on MS medium supplemented 0.6 mg/L BAP + 0.05 mg/L IBA (MS 8). After root, stem, and leaf formation from explants in MS medium, these plants were transferred to soil and grown in the plant growth room for one month. A dependable and effective shoot regeneration procedure was developed, laying the groundwork for genetic transformation in Brassica nigra.

High-frequency shoot regeneration from flower bud derived callus of Gymnostachyum febrifugum Benth., an endemic medicinal plant to the Western Ghats

Gymnostachyum febrifugum Benth. is a small, scapigerous, rare and endemic medicinal herb indigenous to India, and belonging to the family Acanthaceae. This study reports an efficient protocol for high-frequency flower bud-derived callus induction and shoot organogenesis in G. febrifugum. Flower buds at 7 days before anthesis (dBA) were excised from the inflorescence and cultured on MS medium supplemented with various concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D; 2.275–9.1 µM) for callus induction. The optimum callus induction (78%) was obtained on MS medium supplemented with 6.825 µM 2,4-D. The calli when subcultured on MS medium supplemented with different concentrations of thidiazuron (TDZ; 2.275–11.375 µM) or 6-benzylaminopurine (BAP; 2.22–11.1 µM) alone or in combination with 1- naphthaleneacetic acid (NAA; 1.074–3.759 µM) induced shoots. The highest frequency (94%) and number of shoots (44.6 shoots/unit callus) were obtained on MS medium supplemented with 9.1 µM TDZ and 2.685 µM NAA. The optimum rooting frequency (95%) and number of roots (10.2) were observed on ½ MS medium supplemented with 14.76 µM indole-3-butyric acid (IBA). The rooted plantlets were acclimatized and transferred to soil with 94% success. A protocol for high-frequency shoot induction from flower bud derived calli has been standardized for G. febrifugum,an endemic medicinal herb to the Western Ghats.

Indirect Organogenesis from Stem Derived Callus of Dregea Volubilis (L.F) Benth

In vitro propagation methods offer a powerful tool for germplasm maintenance and multiplication. This report established the methodology for callus regeneration of Dregea volubilis from stem explants. The effect of various concentrations and combinations of IAA, NAA, 2,4-D, BAP, KIN and IBA were tested for in vitro callus induction, plant regeneration, and rooting of D. volubilis. The maximum number of greenish, organogenic callus was achieved on the MS medium containing 1.5 mg/L 2,4-D+ 0.5 mg/L KIN and then subsequent shoot regeneration was obtained on MS medium supplemented with 3.0 mg/L KIN from stem derived callus. The highest frequency of shoot regeneration (81.93%) with the maximum number of shoots (10.56 + 0.44) was achieved in this medium. In vitro raised micro shoots showed a 90% response with the maximum number of roots (5.31 ± 0.66) per shoot on MS medium supplemented with 2.5 mg/L IBA after 15 days. Then in vitro rooted plantlets were moved to mud pot bearing soil and vermiculite mixture in 1:1 ratio for hardening and hardened plantlets showed 90% survivability in field conditions. Using this protocol, it is possible to clonally produce viable, uniform and healthy plants with a maximal survival rate that can be used for large scale cultivation, genetic transformation and biopharmaceuticals applications.

Development of Interspecific Hybrids (Abelmoschus Esculentus × A. Tetraphyllus) in Okra Using Embryo Rescue Approach

Undomesticated related species are the rich stock of genes providing resistance to various diseases, pests and unfavourable environmental conditions. The sexual incompatibilities are bottleneck in introgression of such genes from wild species to popular varieties. The present study was executed to standardize a protocol of embryo rescue so to obtain the hybrids of Abelmoschus esculentus × A. tetraphyllus IC141017. Crosses were made between four (Arka Anamika, Pusa Makhmali, Parbhani Kranti, Jammu Okra-05) cultivated varieties of okra (A. esculentus) and a wild species (A. tetraphyllus IC141017) in reciprocal manner. Out of the four popular cultivars used in crossing, fruit set was recorded in Arka Anamika, Pusa Makhmali, Parbhani Kranti only when A. tetraphyallus IC141017 was used as a male parent. Fruit showed distal cracking after 25 d of pollination. The seeds appeared healthy for up to 15 d after pollination and later shrivelled and became pale yellow. Cross combinations, viz., Parbhani Kranti × A. tetraphyllus IC141017, Arka Anamika × A. tetraphyllus IC141017 and Pusa Makhmali × A. tetraphyllus IC 141017 showed the highest shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. Greatest number of shoots per explant (0.92±0.12) as well as the highest frequency of shoot regeneration (86±0.12%) was reported in Parbhani Kranti × A. tetraphyllus IC141017. Arka Anamika × A. tetraphyllus IC 141017 (86±0.12%) and Pusa Makhmali × A. tetraphyllus IC 141017 (82±0.13%) resulted in high frequency of shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. It can be inferred that MS media containing on 0.5 mg l-1 NAA + 1.0 mg l-1 IBA showed positive results in regeneration of interspecific hybrids of okra.

Development of Interspecific Hybrids (Abelmoschus Esculentus × A. Tetraphyllus) in Okra Using Embryo Rescue Approach

ABSTRACT: Undomesticated related species are the rich stock of genes providing resistance to various diseases, pests and unfavourable environmental conditions. The sexual incompatibilities are bottleneck in introgression of such genes from wild species to popular varieties. The present study was executed to standardize a protocol of embryo rescue so to obtain the hybrids of Abelmoschus esculentus × A. tetraphyllus IC141017. Crosses were made between four (Arka Anamika, Pusa Makhmali, Parbhani Kranti, Jammu Okra-05) cultivated varieties of okra (A. esculentus) and a wild species (A. tetraphyllus IC141017) in reciprocal manner. Out of the four popular cultivars used in crossing, fruit set was recorded in Arka Anamika, Pusa Makhmali, Parbhani Kranti only when A. tetraphyallus IC141017 was used as a male parent. Fruit showed distal cracking after 25 d of pollination. The seeds appeared healthy for up to 15 d after pollination and later shrivelled and became pale yellow. Cross combinations, viz., Parbhani Kranti × A. tetraphyllus IC141017, Arka Anamika × A. tetraphyllus IC141017 and Pusa Makhmali × A. tetraphyllus IC 141017 showed the highest shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. Greatest number of shoots per explant (0.92±0.12) as well as the highest frequency of shoot regeneration (86±0.12%) was reported in Parbhani Kranti × A. tetraphyllus IC141017. Arka Anamika × A. tetraphyllus IC 141017 (86±0.12%) and Pusa Makhmali × A. tetraphyllus IC 141017 (82±0.13%) resulted in high frequency of shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. It can be inferred that MS media containing on 0.5 mg l-1 NAA + 1.0 mg l-1 IBA showed positive results in regeneration of interspecific hybrids of okra.

Direct regeneration and genetic fidelity analysis of regenerated plants of Andrographis echioides (L.) - An important medicinal plant

There are several studies about the phytochemical investigations and pharmacological activities of Andrographis echioides (L.), but there is scanty information about its in vitro propagation. Hence the present study is undertaken for in vitro micropropagation of Andrographis echioides (L.) through direct organogenesis. Initially, aseptic leaf explants of in vitro seedlings were excised and inoculated on to plant growth regulators (PGR’s) supplemented in the Murashige and Skoog (MS) media. Among the individual and combinations of PGR’s tested, the combination of thidiazuron (TDZ) along with indole-3-acetic acid (IAA) at 0.5 and 0.2 mg/L concentrations respectively, was proved to be best for high frequency (24.48 ± 1.54) of direct shoot regeneration of A. echioides. Rooting of in vitro raised shoots with the highest rooting percent of 90.42 was carried on IAA (1.0 mg/L), and indole-3-butyric acid (IBA) (1.0 mg/L) amended MS media. in vitro plantlets of A. echioides were hardened in poly cups possessing a 1:2 ratio of compost and garden soil. Initially, hardened plantlets were maintained in a greenhouse for four weeks by irrigating with half-strength MS salt solution and later transferred to field conditions with 80 % survivability. Genetically clonal nature of regenerated plantlets was confirmed by carrying polymerase chain reaction (PCR) based molecular analysis using inter simple sequence repeats (ISSR) and ribulose bisphosphate carboxylase large chain (rbcLa) primers.

In vitro evaluation to intensify the differential morphogenetic response through plant growth regulators and antibiotic supplementation in sugarcane

Sugarcane has always witnessed a lower multiplication rate following conventional method of adoption for newly released variety on large scale by farmers. Tissue culture derived techniques proved as effective, reliable and alternative strategy for rapid production of quality planting materials. In vitro morphogenetic responses varies at genotype basis. Thus, an in vitro investigation was conducted to intensify the differential morphogenetic response through plant growth regulators and antibiotic supplementation in two sugarcane genotypes viz. Co 05011 and Co 0118. The cultures were initiated by explant inoculation on basal MS media and further transferred on different media formulations modified with BAP, Kn, TDZ and GA3. The best morphogenetic response was observed in media M21 [1.5 mg/L (BAP and Kn), 0.25 mg/L (TDZ and GA3)] in genotype Co 05011 whereas, genotype Co 0118 showed the best in media M22 [1.5 mg/L (BAP and Kn), 0.25 mg/L TDZ and 0.5 mg/L GA3]. The higher culture survival percent with sterile shoots was observed in 0.05% Cefotaxime fortified media. Findings of study suggests TDZ as an effective PGR for rapid and high frequency shoot regeneration and the supplementation of 0.05% Cefotaxime in media might enhance sterile culture percent.

Direct shoot regeneration of three Petunia cultivars

A tissue culture system for acquiring high efficiency regeneration of Petunia was optimized. Leaf explants of Alvan, Large Flower Alvan (LF Alvan) and Mahalat cultivars of Petunia hybrida were cultured separately on MS medium including various concentrations of TDZ and BA without auxin in order to assess direct shoot regeneration. Alvan showed the highest frequency of shoot regeneration (100%) and the highest mean number of shoots per explant (25.33) on MS containing 2 mg/l TDZ. For LF Alvan cultivar the highest percentage of shoot organogenesis (100%) and the highest mean number of shoots per explant (18.20) were observed when MS medium containing 1 mg/l BA was used. With the Mahalat cultivar the maximum rate of direct regeneration was obtained on MS supplemented with 0.5 and 1 mg/l BA (80%). The mean number of shoots per explant (9.63) was obtained when 2 mg/l TDZ was used. Regenerated shoots were successfully elongated (2 to 3 cm in length) and transferred into half-strength MS as the rooting medium supplemented with 0.1 mg/l NAA. The shoots were successfully rooted, acclimatized and transferred to the greenhouse.

In vitro Propagation of Indian Teak (Tectona grandis L.) from leaf explants

Efficient plant regeneration via direct organogenesis was established using leaf explants of Tectona grandis L. were tested for growth and organogenic capacity on MS medium fortified with various concentrations of 6-Benzyl Amino Purine and Kinetin (0.5–3.0mg/L) either singly or in various combinations of IAA/IBA (0.5–3.0 mg/L). The highest shoot regeneration frequency (100%) was obtained from leaf explants on MS medium fortified with (1.5 mg/L) IBA/IAA in combination of (2.0mg/L) BAP. At the end of 6 weeks the regenerated shoots were transferred on the same medium (MS + 1.5 mg/L IAA/IBA + 2.0 mg/L BAP) for further proliferation and elongation. The regenerated shoots were rooted with high frequency (60%) in MS medium supplemented with (1.5 mg/L) IBA when compared to other auxin IAA. The in vitro raised plantlets were successfully established in green house and transplanted to natural conditions with 70% survival.

Genome size analysis of field grown and tissue culture regenerated Rauvolfia serpentina (L) by flow cytometry: Histology and scanning electron microscopic study for in vitro morphogenesis

An efficient genetically stable regeneration protocol has been established in Rauvolfia serpentina (L) Benth ex. Kurz, an important medicinal plant, used for the treatment of various cardiovascular diseases. Here, in this present study, plants were regenerated through organogenesis (direct and indirect) and embryogenesis pathways. This is the first ever somatic embryo formation report of R. serpentina in liquid Murashige and Skoog (MS) medium from isolated root segments. The genetic fidelity of the regenerated plants was also analysed by flow cytometry. The 2C DNA content of the regenerated plants raised through organogenesis and somatic embryogenesis was measured along with field grown natural plants, which served as donor plant. The estimated 2C DNA levels are 2.08, 1.76 and 1.88 pg in direct, indirect and somatic embryo regenerated plants respectively while in field grown plant it was noted to be 1.94 pg in R. serpentina. This is perhaps the first ever 2C DNA estimation study in tissue culture raised plants along with natural population of Rauvolfia, an immensely important medicinal plant, present in India. We developed improved callus mediated shoot regeneration (indirect) protocol from leaf callus. Highest shoot regeneration percentage (66.10%) with 4.0 shoots / callus mass was noted on 2.0 mg l−1 BAP + 0.5 mg l−1 NAA added MS medium. The shoot tips and nodal explants on 2.5 mg l−1 BAP amended MS medium induced direct shoot buds, with highest shoot regeneration frequency (81.45%) from shoot tips. On 1.0 mg l−1NAA added MS medium maximum numbers of roots (8.87) were noted, indicating the effectiveness of NAA over IBA in rooting. Excised roots were cultured in agitated liquid MS medium and in 1.0 mg l−1 NAA added conditions somatic embryos were formed. The mature somatic embryos germinated on 1.5 mg l−1 BAP + 0.25 mg l−1 GA3 supplemented MS medium. The histology and scanning electron microscopic (SEM) evidences have been presented to describe the in vitro morphogenesis process. The well-rooted plantlets were acclimatized and transferred to soil successfully (79%). The flow cytometric analysis showed no significant variation in nuclear DNA contents, hence was observed to be genetically stable with their genome size similar to field grown Rauvolfia plants.

High frequency regeneration from leaf derived callus and its histological studies in Lepianthes umbellata

ABSTRACTA protocol was standardized for callus initiation and shoots regeneration from leaf explants of Lepianthes umbellata. The explant was cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 6-Benzylaminopurine (BAP), and 1-Naphthalene acetic acid (NAA) for shoot bud initiation and multiplication. High frequency of shoot regeneration and maximum number of shoots per explant were achieved on MS medium fortified with 0.5 mg L−1 NAA and 1.0 mg L−1 BAP. The elongations of shoots were achieved on the same medium supplemented with 1.0 mg L−1 Gibberelic acid (GA3), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA) at different concentrations on half strength MS medium were used for rooting of the micro shorts. Highest efficiency for rooting was observed on MS with 1.5 mg L−1 IBA. Regenerated plantlets were successfully transferred for acclimatization (70%). Histological studies showed that embryoids and shoot buds initiating from the callus masses, that is, indirect organogenesis. Cytological variations were observed in callus cultures.

In vitro callus induction and indirect organogenesis of Mentha piperita (L.) - an aromatic medicinal plant

The effect of different concentrations and combinations of plant growth regulators were tested for in vitro callus induction and indirect plant regeneration of Mentha piperita L. from young leaves and internodal explants. Greenish, organogenic and nodular nature of callus was achieved only from leaf disc explant when cultured on MS medium containing 1.5 mg/l NAA+ 0.2 mg/l BAP and then subsequent plant regeneration was obtained on MS medium supplemented with 2.0 mg/l BAP+0.5mg/l NAA from leaf derived callus. The highest frequency of shoot regeneration (90%) with the maximum number of shoots (15.1±0.11) was achieved in this medium. In vitro raised microshoots showed 90% response with maximum number of roots (16.3±0.23) on 1.0 mg/l IBA after 11-12 days. Then in vitro rooted plantlets were move to polybags bearing soil and vermiculite mixture in 1:1 ratio for hardening and hardened plantlets showed 95% survivability on field conditions.

In vitro propagation of an endemic and endangered medicinal plant Notopterygium incisum

An efficient system in vitro propagation for Notopterygium incisum Ting ex H. T. Chang, an endemic and endangered medicinal plant, was established to address increased demand and germplasm conservation goals. Optimum response in callus induction (CI) was observed on Murashige and Skoog (MS) medium supplemented with 1.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/l 6-benzylaminopurine (BAP), which the induction rate and growth of callus were 84.44% and 0.67 g respectively. The highest shoot regeneration frequency (76.97%) and maximum number of shoots (3.6 shoots per callus) were achieved on MS medium with 1.5 mg/l BAP and 0.2 mg/l naphthalene acetic acid (NAA). Half-strength MS medium supplemented with 0.6 mg/l indole-3-butyric acid (IBA) was determined to be the best rooting medium, resulting in the maximum number of roots (18.6 roots per shoot) and the highest rooting frequency (92.28%). An approximate 83.8% survival rate among the regenerated plantlets was recorded after they were transplanted in the field at an altitude of 3200 m. An HPLC analysis showed that the content of two main chemical constituents, notopterol and isoimperatorin, in the rhizomes of 3-year-old regenerated plantlets was higher (3.84 mg/g and 4.05 mg/g, respectively) than that in commercially marketed crude drugs. This first report of complete regeneration in vitro could provide an alternative method for the rapid, large-scale production and conservation of this valuable, rare, and endangered medicinal plant.

In vitro regeneration of two Populus hybrid clones. The role of pectin domains in cell processes underlying shoot organogenesis induction

An efficient plant regeneration protocol has been established for two commercial Populus hybrid clones, MC (Populus × euramericana) and UNAL (Populus × interamericana). The culture of internode segments on Murashige and Skoog (MS) medium with 0.5 μM α-naphthalene acetic acid (NAA) and 4 μM N6-benzyladenine for 7 weeks (2 weeks in absence of activated charcoal and 5 weeks in its presence) resulted in the highest frequency of shoot regeneration (100 % for MC and 82 % for UNAL). All regenerated shoots longer than 2 cm rooted on half-strength MS medium, independent of the addition of 0.1 μM NAA. Nevertheless, shoots developed better-formed roots in NAA-free medium, which had a positive effect on the acclimatization of plants. In order to know the cellular processes underlying in vitro shoot organogenesis, a histological study was made in UNAL internode-explants. Results revealed that in vitro culture caused swelling around the cut-off zones in all explants, but only those undergoing organogenesis formed proliferation centers under subepidermal cells, which led to formation of bud primordia. Moreover, in vivo tissues and explants with different in vitro response showed different immunolabelling patterns when they were treated with fluorescentmonoclonal antibodies directed to several pectin-polysaccharides of the cell wall. Results allow us to assign a predominant role of homogalacturonan with a low degree of methyl-esterification in the initiation of bud primordia, a role of β-1,4-D-galactan side chains of rhamnogalacturonan-I in the cellular differentiation, ra ole of α-1,5-L-arabinan side chains of rhamnogalacturonan-I and of homogalacturonan with a high degree of methyl-esterification in cell division and growth.

An improved thin cell layer culture system for efficient clonal propagation and in vitro withanolide production in a medicinal plant Withania coagulans Dunal

In vitro micropropagation based on transverse thin cell layer (tTCL), which utilizes a very small number of cells or tissues, has now emerged as a powerful tool in plant tissue differentiation and regeneration. In this study, intense, fast rate shoot multiplication has been achieved utilizing thin cell layer explants for endangered pharmaceutical plant Withania coagulans Dunal (family Solanaceae) having high hypoglycemic potential. To develop an efficient protocol for rapid in vitro propagation of W. coagulans stem node, shoot apical meristem (SAM), and tTCL were used as explants and inoculated in different strength of MS medium. The tTCL explant showed high frequency of shoot regeneration and was also affected by the concentration of plant growth regulators. The full strength solid MS medium was optimal for shoot regeneration (31.94 ± 1.39%) and the highest percent of shoots (93.05 ± 2.77) were observed in MS medium fortified with 2.0 mg l−1 BAP and 0.5 mg l−1 NAA. In contrast to tTCL, SAM cultured on the same medium showed significantly lower multiplication rate with only 1.16 ± 0.08 average shoots per responsive explants. The best rooting of the regenerated shoots was achieved in half strength liquid MS medium supplemented with 2 mg l−1 IBA. Plants were successfully transferred to the field after acclimatization with a survival rate of 90.6%. Monomorphic nature of ISSR and RAPD markers in this study confirmed the genetic fidelity of the in vitro raised tTCL clones. Quantification of withanolides content through high performance liquid chromatography (HPLC) showed 1.4 fold increases in withaferin A content and 1.6 fold withanolide A content in acclimatized field grown plants compared to field grown (wild type) plants. In acclimatized field grown plants the over-expression of SQS gene further attested the improved withanolide production in tTCL micropropagated plants having valuable anti-diabetic potential. The current study reveals the fast rate, cost effective micropropagation and conservation of W. coagulans through tTCL technique for its use as anti-diabetic agent at commercial level.

Efficient direct shoot organogenesis, genetic stability and secondary metabolite production of micropropagated Digitalis purpurea L.

Cardiovascular and cancer diseases are the first causes of death in the world. Digitalis purpurea L. is a medicinal plant that produces secondary metabolites, like digoxin and digitoxin, which are employed in therapies against heart failure. Moreover, anticancer and antiviral properties of these metabolites have recently been described. The present work details a method to obtain in vitro plants of D. purpurea from leaf segments through direct organogenesis. A reliable and efficient plant induction system was established by optimizing the concentration of naphthaleneacetic acid (NAA) and 6-benzylaminopurine (6-BAP). The highest frequency of shoot regeneration (98.5%) with an average number of shoots per leaf segment of 18.9 was achieved via direct organogenesis from leaf segment on MS medium containing 0.54 μM NAA + 13.2 μM 6-BAP. Additionally, Random Amplified Polymorphic DNA (RAPD) analysis showed 100% monomorphic bands, which indicated genetic stability of the obtained plants. Moreover, leaf powder of de novo regenerated plants fulfills the quality specifications of the British Pharmacopoeia and HPLC analysis revealed the presence of digoxin (22.6 μg gDW−1) and digitoxin (220.7 μg gDW−1) without significant differences in contents between de novo regenerated and mother plants. An efficient in vitro propagation protocol via direct organogenesis and genetic stability assessment of D. purpurea for obtaining leaf powder with quality for the use as raw material have thus been described. The protocol also provides an effective means for several approaches in biotechnology and breeding programs, in order to produce pharmaceutically interesting cardenolides.

In vitro Plantlet Regeneration from Callus Culture of Trachyspermum copticum

The effect of explant sources and plant growth regulators on callus induction and plantlet regeneration of Trachyspermum copticum were explored. Different explants including hypocotyl, cotyledonary node and leaf were cultured on MS supplemented with different combinations and concentrations of plant growth regulators including 2,4‐D (0.2‐3 0.5 mg/l), NAA (2 mg/l), BAP (1‐3 mg/l), Kn (0.5 mg/l) and IAA (0.8 mg/l). The best response for callus induction (100%) as well as quality was observed from cotyldonary node segments cultured on MS supplemented with 2, 4‐D at 1 mg/l in combination with Kn at 0.5 mg/l. Calli derived from various explants were subcultured on shoot induction media with different compositions and concentrations of medium. MS without any plant growth regulator promoted the highest frequency of shoot regeneration (100%) and also mean number of developed shoots per explants (3.8) showed the same result. Regenerated shoots were then rooted on three‐fourth strength MS with 75% efficiency after 30 days.Plant Tissue Cult. & Biotech. 27(1): 13-20, 2017 (June)

Improved production of Oroxylum indicum (L.) Kurz by altering the salts of the medium

The present study was conducted to test the effects of KNO3, KH2PO4, and CaCl2 on shoot multiplication, root proliferation, and accumulation of phytochemicals in in vitro cultures of Oroxylum indicum. The results indicate that modifying the MS salt formulation in relation to particular inorganic nutrients highly affected shoot multiplication, root proliferation, and accumulation of flavonoids in in vitro cultures. A concentration of 0.60 g L−1 CaCl2 resulted in the highest frequency of shoot regeneration (5.6 shoots per explant). A concentration of 0.40 g L−1 CaCl2 resulted in the highest frequency of root regeneration (7.8 roots per shoot). Modifications of the concentrations of inorganic salts were also found to be advantageous for production media for both multiple shoots and shoot-derived root in vitro cultures. Multiple shoots generated on shoot induction medium with a concentration of 0.60 g L−1 CaCl2 and roots generated on root induction medium with a concentration of 1.5 g L−1 KNO3 yielded about a five times higher flavonoid level than cultures generated on control medium respectively.