Internode Explants Research Articles

Encapsulation of internode regenerated adventitious shoot buds of Indian Siris in alginate beads for temporary storage and twofold clonal plant production

This study for the first time demonstrates single bead alginate encapsulation and conversion (multiple shootlets rejuvenation) from adventitious shoot buds (AB) of Albizia lebbeck (L.) Benth. Internodal (IN) segments isolated from field grown 1-year-old plant of A. lebbeck were used for AB induction under in vitro conditions. IN segments incubated on MS medium augmented with 10.0 µM BA exhibited highest adventitious shoot bud induction frequencies (76 %) on all over the surface after 10 weeks of culture. Induced AB were detached from in vitro proliferating cultures and used for encapsulation as an explant to produce large number of synseeds (07–08) from a single IN explant. Four to five AB were encapsulated in a single calcium alginate bead to manage mass propagation, interim storage and germplasm sharing. The finer gel matrix for encapsulation was attained using 3.0 % sodium alginate and 100 mM calcium chloride. Highest percentage of shoot emergence and multiplication (75 %) from synseed was obtained on MS + 10.0 µM BA + 1.0 µM NAA (RM) after 10 weeks of culture. Encapsulated adventitious buds stored at 4 °C for 1–8 weeks (2 months) too showed thriving shoot emergence (68 %) and multiplication in encapsulated AB and development into complete plantlets when returned to RM. Hence, 4–5 encapsulated AB stored at 4 °C, when cultured back to RM also showed shoot induction resulting in up to 10 shoots per synseeds after 10 weeks of culture. Healthy root formation (½ MS + 2.0 µM IBA) and acclimatization were optimized by using previously standardized protocol (Perveen et al. in J For Res 22:47–52, 2011). Genetic stability of synseed-derived plantlets acclimatized under ex vitro was assessed and compared with mother plant using inter-simple sequence repeats (ISSR) analysis. The synthetic seeds have the achievability of being a substitute planting material for the forestry sector in future, especially for the multipurpose plant species.

Callus induction and multiple shoot regeneration in Ajuga bracteosa Wall ex. Benth.-An important medicinal plant growing in Kashmir Himalaya

Ajuga bracteosa Wall ex. Benth is an important medicinal plant growing in Kashmir Himalaya. During the present study an efficient and rapid in vitro protocol has been established viz; Callus induction and multiple shoot regeneration. Callus induction has been achieved from different explants viz., leaf, petiole and internodal cuttings. Maximum callus production was obtained when leaf explants were inoculated on Murashige and Skoog (1962) medium supplemented with BAP (benzylaminopurine 5.0 mg/l) after 19 days of inoculation. In the petiole explant callus was induced on MS (Murashige and Skoog ) medium supplemented with BAP (2.0 mg/l) + IAA ( Indole-3-acetic acid 3.0 mg/l) after 51 days of inoculation and from internodal explant callus was induced on MS medium supplemented with BAP (2.0 mg/l) + NAA(α-Naphthalene acetic acid 5.0 mg/l) after 35 days of inoculation. Callus derived from leaf explants differentiated into multiple shoots on MS medium supplemented with different concentrations of auxins (IAA, NAA) and cytokinins (BAP, Kinetin). Maximum multiple shoots regenerated on MS medium supplemented with BAP (5.0 mg/l) after 28 days of inoculation.

An Improved System for Rapid in vitro Regeneration of Saintpaulia ionantha

For rapid multiplication and genetic manipulation of African violets (Saintpaulia ionantha Wendl.) was aimed at developing a rapid and efficient regeneration and adaptation system from leaf explants. Using RM medium supplemented with different combinations of growth regulators, we developed a highly efficient and time-saving in vitro regeneration protocol. The developed system was also successfully applied for plant regeneration from petiole and internode explants of several Saintpaulia cultivars at a high efficiency. Regeneration occurred through both somatic embryogenesis and shoot organogenesis at a high frequency. Also, we developed a rapid root formation protocol followed by their efficient adaptation for in vitro regenerated plantlets. Plant Tissue Cult. & Biotech. 24(1): 37-45, 2014 (June) D. O. I. http://dx.doi.org/10.3329/ptcb.v24i1.19194

Somatic embryogenesis and plant regeneration in Wedelia calendulacea Less. an endangered medicinal plant

In this work, plant regeneration via somatic embryogenesis was achieved from leaf and internode derived callus of Wedelia calendulacea, an endangered medicinal plant. Primary callus was induced by culturing leaf disc and internode explant on Murashige and Skoog medium supplemented with 2.0 mg L-1 of 2,4-D under light condition. Transfer of embryogenic callus on a reduced concentration of 2,4-D facilitated somatic embryo development while calluses remained unorganized at the same 2,4-D level. A histological analysis confirmed somatic embryo by revealing the presence of a closed vascular system in the developing embryos and lack of a vascularconnection with surrounding callus tissues. Somatic embryos germinated into plantlets upon transfer on MS medium containing 1.0 mg L-1 BAP plus 0.5 mg L-1 GA3. Plantlets were acclimatized successfully and survived under soil condition. This is the first on somatic embryogenesis of W.calendulacea. This result could facilitate genetic transformation of this important medicinal plant.

In Vitro Induction of Flower Mutation in Catharanthus roseus Using Gamma Irradiation

The purpose of this study was to study the effect of gamma irradiation and in vitro culture on mutation induction of flower color in Catharanthus roseus. A number of plants were regenerated from irradiated internode explant. Tow cultivars from previous study Rosea and Alba, and four doses of gamma irradiation (0.0, 0.5, 0.75 and 1.0 Gy) were applied. Obtained results indicated that both 0.75 and 1.0 Gy irradiation doses were the most effective in inducing mutation in flower shape and color, while the dose of 0.5 Gy was effective in inducing mutation in leaf form.

In Vitro Regeneration and Mass Multiplication of Prunella vulgaris L.

This is the first report on the development of an in vitro regeneration protocol for the medicinally important plant Prunella vulgaris L. growing in Doda region of Jammu and Kashmir. A simple, high-frequency and reproducible protocol has been standardized. Callus induction from different explants was obtained on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) and 2,4-dichlorophenoxy acetic acid (2,4-D) in various concentrations. However, the best response towards callus formation with internode explants was observed in the medium having 1.0 mg l−1 TDZ and 0.5 mg l−1 2,4-D. The callus thus obtained proliferated on MS medium containing 1.0 mg l−1 TDZ and 0.05 mg l−1 indole-3-acetic acid (IAA). The green nodular callus led to the formation of shoots on MS medium supplemented with 1.5 mg l−1 benzyl amino purine (BAP) and 0.01 mg l−1 IAA. The shoots from the nodal explants were observed with 0.5 mg l−1 BAP and 0.05 mg l−1 IAA on MS medium. The best rooting response was observed with 0.5 mg l−1 IBA. The regenerated plants were transferred to the field after acclimatization.

High Frequency Plant Regeneration from Leaf Derived Callus of <i>Dianthus caryophyllus</i> L.

An efficient procedure was developed for in vitro callus induction, proliferation and regeneration of carnation cultivar (Dianthus caryophyllus L.) using leaf, nodal and inter-nodal explants on Murashige and Skoog’s medium (MS) supplemented with exogenous plant growth regulators. For morphogenic callus induction and proliferation from various explants, MS medium supplemented with 3.0 mg/l 2,4-D was highly efficient with 100% callus induction frequency from inter-nodal explants. Leaf explants showed quicker response than nodal and inter-modal explants, for callus initiation within 6 days of inoculation. Best grown callus was obtained from leaf explant. The leaf-derived callus was maintained up to several weeks, which indicated that 8-week incubation period was the most suitable for obtaining well proliferated, morphogenic callus. Temperature variation also affected the growth of in vitro induced morphogenic callus from various explants. Results have shown that 27°C proved to be the best temperature for morphogenic callus induction and proliferation from leaf and inter-nodal explants. Among the auxin-cytokinin combination, MS medium containing 1.0 mg/l N(6)-benzylaminopurin (BAP) and 2.0 mg/l NAA showed the highest efficiency of callus initiation and proliferation from leaf, nodal and inter-nodal explants. Light conditions proved better for callogenesis and proliferation from leaf, nodal and inter-nodal explants. Regeneration response from well grown morphogenic callus was prominent on MS medium supplemented with 3.0 mg/l BAP alone and 1.0 mg/l NAA with 3.0 mg/l BAP.

In vitro chemical mutagenesis in exclusively vegetatively-propagated rose-scented geranium (Pelargonium spp.)

SummaryRose-scented geranium (Pelargonium spp.) is an important, vegetatively-propagated, aromatic plant. In such plants, genetic variation may be generated through somaclonal variation and/or induced mutagenesis. Internode and petiole explants of two clones, ‘Bourbon’ and ‘Narmada’, were treated with 0, 0.25, or 0.60 mM N-nitroso-N-methylurea (NMU) to generate variations in oil yield, oil components, and to study the effectiveness of selection for herbage yield and oil concentration. Somaclones were regenerated and evaluated for six oil yield components in the SC2 generation (i.e., the second vegetative generation after the in vitro propagation phase) and in the M1V2 generation (i.e., the second vegetative generation after in vitro mutagen treatment). Significant variations were observed in the majority of the oil yield components studied following all three NMU treatments. Of the 36 somaclones selected for higher herbage yields or oil concentrations in the SC2 and M1V2 generations, 14 and 15 somaclones exhibited significantly higher herbage yields and oil concentrations, respectively, and gave 38 – 137% higher oil yields than their respective parental clones in the next vegetative generation. As only one somaclone with a higher oil yield than the parental clone was obtained from the 0 NMU treatment, in vitro mutagenesis with NMU could be more useful than somaclonal variation for genetic improvement in rose-scented geranium.

Cultivo in vitro de Brosimum gaudichaudii Tréc. (Moraceae)

O objetivo desta pesquisa foi avaliar o potencial organogenético de entrenós, discos foliares, e raízes de Brosimum gaudichaudii utilizando 12 diferentes combinações dos fitorreguladores 6-benzilaminopurina e ácido naftaleno acético, em meio MS (diluído à metade), sólido (6,5 g de ágar), e com 20 g.L-1 de sacarose. Independentemente das combinações hormonais testadas verificou-se a formação de calos friáveis (2 a 20 mm de diâmetro) em 90% dos entrenós usados como explantes. No entanto, os tratamentos testados não foram capazes induzir calos ou gemas em raízes e em discos foliares. O estudo anatômico revelou a formação de meristemóides nas regiões mais externa e mais interna dos calos. Os resultados obtidos poderão servir de base para novos testes de indução de calos na espécie.

In Vitro Regeneration and Somaclonal Variation of Catharanthus roseus Don. Using Leaf and Internodal Explants

In Vitro Regeneration and Somaclonal Variation of Catharanthus roseus Don. Using Leaf and Internodal Explants

Production and optimization through elicitation of carotenoid pigments in the in vitro cultures of Cleome rosea Vahl (Cleomaceae)

The present study is the first account of in vitro carotenoid pigment induction in Cleome rosea, a Brazilian herbaceous species frequently found near the coast in ecosystems submitted to intense anthropic degradation. Micropropagated plants obtained from in vitro roots were used as source of internodal explants for callus cultures. The callus cultures were induced on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram) in the light or in the dark. The use of different culture media (B5, Nitsch and White) supplemented with 2,4-D was also evaluated. Although callogenesis was obtained in all treatments, a high production of biomass was achieved from the cultures that were maintained in light. The highest biomass accumulation was reached by cultures established on MS medium supplemented with 0.2 mg L−1 2,4-D. The exposure of cultures to light was an essential factor for carotenoid production. Pigment induction was observed on calli maintained in all tested media, and the highest pigment yield was obtained by cultures established on MS medium with 0.2 mg L−1 2,4-D. Callus cultures were subjected to treatments with elicitors (chitosan, methyl jasmonate, and yeast extract) at different concentrations and exposure times. The highest pigment production was achieved on cultures treated with 60 mg L−1 methyl jasmonate (MeJa), which resulted in a six-fold increase in the carotenoid yield when compared to non-elicited cultures. A chromatographic analysis showed that the addition of MeJa induced β-carotene production. Elicited and non-elicited calli were used to establish cell suspension cultures. These cultures were evaluated during three subsequent subcultures, showing an increased production of carotenoids during the first subculture. This study showed that in vitro cultures of C. rosea, especially after elicitation, may become an efficient alternative source of carotenoids indicating the success of plant tissue culture techniques for secondary metabolite production.

Organogenesis from in vitro-derived leaf and internode explants of Hoya wightii ssp. palniensis -a vulnerable species of Western Ghats

An efficient system was developed for indirect plant regeneration from in vitro-derived leaf and internode explants of Hoya wightii ssp. palniensis. Maximum percentage of the organogenic callus was obtained on MS medium supplemented with NAA (1.0 mg/l) and 2,4-D (2.0 mg/l). The best shoot bud induction was observed on MS medium with BA (1.0 mg/l) +IBA (0.5 mg/l). The coconut water (15%) was better, resulting in a differentiation of the shoot initials in to well-developed shoots. The elongated shoots (› 3cm long) were rooted on a full strength MS basal medium, supplemented with 0.2 mg/l of IBA. Finally, the rooted plants were transferred to the soil with 80% success rate. This protocol was utilized for the in vitro propagation of this endangered plant species.

Efficient plant regeneration of yellow loosestrife (Lysimachia vulgarisL.), a medicinal plant

Lysimachia vulgaris L. (yellow loosestrife) is a medicinal plant that has been used in the treatment of fever, ulcer, diarrhea and wounds in traditional medicine. A reliable in vitro culture protocol for yellow loosestrife was established. Explants (leaf lamina, stem internode and root segments) were cultured on Murashige and Skoog minimal organics (MSMO) medium supplemented with various plant growth regulator combinations. Of the tested combinations, those involving benzyladenine (BA) with either indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) were the most effective for all used explants in shoot production. Best shoot proliferation was obtained from leaf lamina explant cultured on media containing 0.5 mg/l BA and 0.1, 0.5 or 1 mg/l IBA, from stem internode explant cultured on media containing 1 mg/l BA and 0.5 mg/l IBA or 0.01 mg/l thidiazuron (TDZ) and 0.5 mg/l IAA, and from root explant cultured on media containing 0.5 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were rooted on MSMO medium containing different concentrations of IAA, IBA, 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA). IBA was determined as the most effective auxin for rooting. Most shoots developed roots on medium with 0.5 mg/l IBA.

In vitro Regeneration and Agrobacterium-mediated Transformation of Potato (Solanum tuberosum L.) Cultivars Grown in Mexico

Prior to Agrobacterium-mediated genetic transformation in vitro regeneration protocol was established for three potato cultivars (Alfa, Cambray Rosa Morelos and Atlantic) grown in Mexico using leaf, node and internodal explants. Regeneration protocol was developed with or without the intervention of callus. Two potato cultivars, namely, Cambray Rosa Morelos and Alpha were transformed using Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121 containing the GUS and nptII genes. GUS histochemical assay and PCR analysis were conducted on rooted shoots grown in media without hormones but supplemented with antibiotics. Transformed shoots tested positive through GUS histochemical assay and integration of nptII gene was confirmed by PCR analysis DOI: http://dx.doi.org/10.3329/ptcb.v22i2.14193 Plant Tissue Cult. & Biotech. 22(2): 93-105, 2012 (December)

In vitro organogenesis from leaf and transverse thin cell layer derived callus cultures of Talinum triangulare (Jacq.) Willd.

Talinum triangulare is an important medicinal herb used traditionally in the treatment of various diseases. The present study was intended to develop a rapid and efficient protocol for indirect organogenesis from leaf discs and transverse thin cell layer (tTCL) of internodal explants of T. triangulare. Best callusing response (100 %) was observed with tTCL explants on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyl amino purine and 5.37 μM α-naphthalene acetic acid (NAA). High frequency shoot regeneration (96.67 %) was obtained from tTCL derived calli on MS medium supplemented with a combination of 0.45 μM thidiazuron and 0.27 μM NAA, by producing 9.20 ± 0.35 shoots with a shoot length of 2.74 ± 0.03 cm. In vitro rooting of the microshoots was recorded on half-strength MS medium containing 2.46 μM indole-3-butyric acid by eliciting 15.20 ± 0.27 roots with a length of 4.25 ± 0.11 cm. The rooted shoots were acclimatized on garden soil, sand and coco pith (1:1:3 v/v) planting substrate. The plantlets were successfully established under field conditions with 100 % survival rate. The hardened plants exhibited homogeneity and no observable morphological variations were detected among the regenerants and the mother plants of T. triangulare.

Micropropagation ofHibiscus sabdariffaLinn. with reference to anthocyanin production in callus

An effective protocol for in vitro propagation from leaf, node and internodal explants of Hibiscus sabdariffa L. was achieved by organogenesis. Two morphologically distinct type of calli were obtained in different combinations and concentrations of auxins and cytokinins. Creamy yellowish friable calli were obtained when MS medium was supplemented with 2,4-Dichlorophenoxy acetic acid (2,4-D), Napthalene acetic acid (NAA) and 6-Benzylamino purine (BAP). Compact green organogenic calli were obtained when MS medium was supplemented with various concentrations of NAA and BAP. Shoot formation was obtained directly from nodal explants when MS medium supplemented with various concentration of NAA. Calli derived from various explants such as leaf, node and internode showed induction of anthocyanin pigments. MS medium supplemented with 2,4-D+BAP (0.5+2.0 mg/L) and 6% sucrose showed intense pink colour anthocyanin pigmentation.

A dark incubation period is important for Agrobacterium-mediated transformation of mature internode explants of sweet orange, grapefruit, citron, and a citrange rootstock.

BackgroundCitrus has an extended juvenile phase and trees can take 2–20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees.Methodology/Principal FindingsFactors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings.SignificanceThe critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.

Agrobacterium-mediated Genetic Transformation of Chrysanthemum (Chrysanthemum morifolium Ramat.) with an Aphidicidal Gene, gcs (Gamma-cadinene Synthase)

Florist’s chrysanthemum (Chrysanthemum morifolium Ramat.) belongs to the Asteraceae family and represents the second most important floricultural crop in the world. Unfortunately most genotypes are sensitive to aphids. Internode explants of 1581 and 4043 genotypes were incubated with A. tumefaciens strain AGL-0 containing pBIN plasmid with the npt gene as a selectable marker for kanamycin resistance and gcs gene as an aphicidal gene with rbcS promoter. Kanamycin resistant shoots were induced from internodes after 3 weeks. Finally, the shoots were rooted on MS medium containing 30 mg L-1 kanamycin. Incorporation and expression of the transgenes were confirmed by PCR and RT-PCR analysis. Genotype 4043 has been transformed in this study for the first time. Transformation frequency for GCS was 6.25 and 5% for genotypes 1581 and 4043, respectively.

A Somaclonal Variant of Rose-Scented Geranium (Pelargonium spp.) with Moderately High Content of Isomenthone in its Essential Oil

Rose-scented geranium (Pelargonium spp.), which is highly valued for its essential oil, is exclusively propagated vegetatively. Hence no genetic improvement work is possible through conventional breeding. Somaclonal variation was generated with and without in vitro mutagenesis using N-nitroso-N-methyl urea (NMU) in an Indian cultivar 'Bourbon', and a clone 'Narmada'. A somaclonal variant (N75) with a moderately high content of isomenthone in its essential oil was isolated from somaclones generated after treatment of internodal explants of clone, 'Narmada' with 0.25 mM NMU for 1 h. The contents of isomenthone in its essential oil were 26% and 35%, respectively, in SC2/VM2 and SC3/VM3 generations (second and third vegetative generations, respectively, after in vitro mutagen treatment) as compared with 0.7% and 0.3%, respectively, in the parental clone, 'Narmada'. The contents of alcohols and their esters (linalool, citronellol, geraniol, citronellyl formate and geranyl formate) in the essential oil of N75 in SC2/VM2 and SC3/VM3 generations were 49% and 35%, respectively, as compared with 69% and 63%, respectively, in the parental clone, 'Narmada'. This is the first report on a chemovariant of rose-scented geranium with a moderately high content of isomenthone. All earlier reported isomenthone-rich variants of rose-scented geranium had quite high contents of isomenthone (64-71%) in their essential oils. The probable modes of origin of this somaclonal variant, its parental clone 'Narmada' (with very low content of isomenthone) and four earlier reported isomenthone-rich variants of Indian cultivars of geranium are discussed.

Standardization of Protocol for Agrobacteriummediated Transformation in Potato (Solanum tuberosum L.)

The experiment was conducted at the Laboratory of Biotechnology, Biotechnology Division, Bangladesh Agricultural Research Institute, Gazipur- 1701 during July 2007 to June 2008. An efficient and reproducible protocol for the production of transgenic potato plants was developed by inoculating internode explants of potato with Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBI121 having one reporter gene (gus) and selectable marker gene (nptII) resistant to Kanamycin. The transformation experiment was done by optimizing two important parameters names infection time and cocultivation period. Most of the explants produced shoots within 21 days on 5 mg/l Zeatin riboside (ZR) and 50 mg/l Kanamycin supplemented MS medium without introducing callus. The infected explants produced 8.27 and 6.42 shoots in Asterix and Diamant varieties, respectively within 21 days. Transgenes were confirmed by molecular analysis. DNA from well established rooted plants confirmed nptII positive through PCR analysis. The transformation rates were 28.97 and 24.37% in Asterix and Diamant, respectively. Putative transformed plants of Diamant and Asterix varieties produced roots in ½MS medium supplemented with 50/mg Cefotaxim, 50 mg/l Kanamycin and 0.5 mg/l IBA. DOI: http://dx.doi.org/10.3329/bjar.v37i2.11220 Bangladesh J. Agril. Res. 37(2): 185-194, June 2012