Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

Improved in vitro clonal propagation of Rauwolfia serpentina L. Benth – An endangered medicinal plant

In this protocol, 96% of bud proliferation was observed after 14 days of inoculation. Highest rate of shoot multiplication (13.3 shoots per explant) with an average shoot length of 8.2 cm was observed in Murashige and Skoog (MS) medium+2 mg/l 6-benzylaminopurine+0.25 mg/l 1-naphthaleneacetic acid+3% sucrose. Half MS (1/2 MS)+0.5 mg/l indol-3-butyric acid proved best with 73.3% rooting. The rooted shoots showed 90% survival. Genetic uniformity of the micropropagated plants with their donor plants was confirmed through random amplified polymorphic DNA molecular technique.     

In vitro culture of Capparis decidua and assessment of clonal fidelity of the regenerated plants

A protocol for in vitro multiplication of Capparis decidua (Forsk.) Edgew. has been developed from cultured leaves procured from multiplying axillary shoots on the cultured nodal explants. The highest efficiency of shoot formation was observed on Murashige and Skoog (MS) medium containing 2 mg dm⁻³ benzyladenine (BA) and 0.5 mg dm⁻³ 1-naphthaleneacetic acid. The regenerated shoots were transferred to MS medium containing 3 mg dm⁻³ BA for growth and proliferation. Shoots above 2 cm in length were transferred to MS medium supplemented with 1 mg dm-3 indole-3-butyric acid plus 0.5 mg dm⁻³ indole-3-acetic acid for root induction. No variation was detected among the micropropagated plants by randomly amplified polymorphic DNA (RAPD) markers.     

Rapid clonal propagation of three mulberries,Morus cathayana HemsL,M. lhou Koiz. andM. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees

High-frequency bud break and multiple shoots were induced in apical shoot buds and nodal explants ofMorus cathayana, M. lhou andM. serrata on Murashige and Skoog (MS) medium containing 0.5–1.0 mg/l 6-benzylaminopurine (BAP). Addition of gibberellic acid (0.4 mg/l) along with BAP induced faster bud break both in apical shoot buds and nodal explants and also enhanced the frequency of bud break in all three species. Shoot culture initiation was greatly influenced by explant type, explant age and explanting season. The shoots were successfully rooted on half-strength MS medium containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/l. The plantlets were successfully acclimated and eventually established in soil.Abbreviations BAP 6-Benzylaminopurine - GA ₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthalene acetic acid     

In vitro propagation of herbaceous peony (paeonia lactiflora pall.) by a longitudinal shoot-split method

A procedure for the clonal propagation ofPaeonia lactiflora Pall. cvs. Takinoyosooi and Sarah Bernhardt through shoot tip culture is described. Half strength Murashige and Shoog (1962) medium supplemented with 0.5 mg/l 6-benzylaminopurine plus 1 mg/l gibberellic acid promoted formation and growth of axillary buds. Continuous shoot multiplication was achieved by vertically splitting the shoot axis and subsequent division of elongated axillary shoots every 36 days. High frequency (57–100%) of rooting was obtained on paper-bridge liquid medium supplemented with 1 mg/l indole-3-butyric acid. Half of the rooted plantlets were established on porous soil. Thus, 700 and 300 plants of cv. Takinoyosooi and Sarah Bernhardt could be theoretically obtained from a single bud in one year.Abbreviations BAP 6-benzylaminopurine - GA gibberellic acid - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) basal medium     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil)

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA₃) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Micropropagation of meadowfoam (Limnanthes spp.)

Summary A rapid micropropagation system was developed for meadowfoam (Limnanthes spp. Brown) using four genotypes of three species. Murashige and Skoog (MS) medium supplemented with N⁶ benzyladenine (BA) and indole-3-acetic acid (IAA) at 0, 0.1, 0.5, 1.0 and 2.0 mg/l was tested for multiplication, shoot elongation and rooting. Expiants were taken from pot-grown plants. The most useful level for shoot growth and multiplication of both floral induced and non-induced plants was 0.5 mg/l BA. IAA failed to affect shoot growth or multiplication. Expiants from non-induced plants multiplied at moderate to high rates on 0.5 mg/l BA, while those from induced plants multiplied slowly and tended to elongate and flower. Non-induced plants on 2 mg/l BA produced large numbers of tiny shoots; induced plants did not respond. Shoots of all genotypes rooted on MS medium without hormones and all plants grew normally after transplanting to soil. This system provides a new tool for the development of meadowfoam as a crop plant.Abbreviations (BA) N ⁶ -benzyladenine - (IAA) indole-3-acetic acid - (MS) Murashige and Skoog medium, 1962     

Micropropagation of a fruit tree, Morus australis Poir. syn. M. acidosa Griff.

High frequency bud break and multiple shoots were induced in nodal explants collected between November to February from a 5 year old tree of Morus australis Poir syn. M. acidosa Griff. on Murashige and Skoog's medium supplemented with 6-benzylaminopurine (1.0 mg/1). Incorporation of gibberellic acid (0.3 mg/l) along with BAP (1.0 mg/l) not only induced faster bud break from nodal explants as well as from apical shoot buds, but it also enhanced the frequency of bud break. Nodal explants were more responsive than apical shoot buds. The shoots formed in vitro were multiplied further as nodal segments, and an average multiplication rate of 6-fold per subculture was established within 4–5 months. The shoots were successfully rooted on half-strength MS containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/1. The plantlets were successfully hardened off and established in natural soil.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - KN kinetin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA indole-3-propionic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalene acetic acid     

Somatic embryogenesis and plant regeneration in Quercus acutissima

Immature embryos of Quercus acutissima were collected weekly beginning 5 weeks post-fertilization and cultured on modified MS(Murashige and Skoog) medium containing 1,000 mg/l glutamine and 5 mM proline with different combinations of IBA(0.5–10.0 mg/l) and BA(0 or 1.0 mg/l) in light. The highest percentage of embryogenic cultures occurred on the medium containing 0.5 mg/l IBA or 1.0 mg/l BA and 0.5 mg/l IBA. Four weeks after initiation, the embryogenic cultures were transferred to MS medium without plant growth regulators and cultured for 4 weeks. The somatic embryos were then transferred to germination medium. The best germination results were achieved from WPM(Woody Plant Medium) containing 0.1 mg/l BA. Plantlets from somatic embryos were incubated on WPM supplemented with 0.2 mg/l BA for 4 weeks and plantlets with well developed shoots and roots were transplanted to perlite and peat moss(11, v/v) mixtures and placed in a culture room. After being hardened off for 8 weeks, they were transferred outdoors where they grew.Abbreviation BA N⁶-benzyladenine - IBA indole-3-butyric acid - GA₃ gibberellic acid - ABA abscisic acid - MS Murashige & Skoog Medium - WPM Woody Plant medium     

In vitro propagation of shoot buds of Carica papaya L. (caricaceae) var. Honey Dew

Shoot buds from the saplings and the fruit bearing plants of Carica papaya L.. var. Honey Dew (papaya) initially treated with Gentamycin were cultured in modified MS media, each with a different hormonal combination, for the establishment of cultures and multiplication and rooting of plants. About 43% of explants from fruit bearing plants and 69% of those from saplings remained free of contamination and retained regeneration capacity when treated in 500 mg/l Gentamycin. For the establishment of the explants a medium containing 1 mg/l GA₃ and 2 mg/l kinetin was necessary. When established buds were transferred to medium containing 1 mg/l NAA and 3 mg/l kinetin, calli were initiated at cut ends of shoot buds; multiplication started on transfer to NAA (0.1 mg/l) and BAP (0.5 mg/l) medium. Cultures have been maintained for the last twenty months without any loss in multiplication rate. Rooting was induced in medium with reduced salt concentration containing 2 mg/l IBA. Shoot elongation was induced after prolonged culture in the same rooting medium.Abbreviations MS Murashige and Skoog, 1962 - SH Schenk and Hildebrandt, 1972 - GA₃ Gibberellic acid - Kn Kinetin - NAA Napthaleneacetic acid - BAP 6 -Benzylaminopurine - IBA Indole-3-butyric acid - IAA Indole-3-acetic acid     

Prolific shoot regeneration of <Emphasis Type="Italic">Astragalus cariensis</Emphasis> Boiss

Prolific shoot regeneration via organogenesis was induced from leaf and leaf petiole explants of the endemic Astragalus cariensis species on Murashige and Skoog (MS) medium with α-naphthaleneacetic acid (NAA) and benzyladenine (BA) within 8 week. The highest number of shoots (23/explants) was obtained from leaf explants cultured on MS with 0.5 mg/l NAA and 4 mg/l BA. Elongated shoots were successfully rooted in MS medium with 0.5 mg/l indole-3-butyric acid. Rooted plantlets were acclimatized in pots containing 1:1 mixture of peat and perlite.     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

In vitro somatic embryogenesis from callus culture of the timber yielding treeHardwickia binata Roxb.

Somatic embryogenesis was achieved in callus cultures derived from immature cotyledonary explants ofHardwickia binata Roxb., a multipurpose leguminous tree, on semisolid modified Murashige and Skoog's (mMS) medium containing 2900 mg/l potassium nitrate (KNO₃) supplemented with 4.64 µM kinetin (Kn) and 5.37µM a-naphthaleneacetic acid (NAA). Somatic embryos proliferated rapidly after transfer to MS basal medium supplemented with 2052.6 µM L-glutamine and 0.084 µM gibberellic acid (GA₃). Maturation of somatic embryos was achieved on half-strength MS basal medium supplemented with 1.23 µM IBA and 2% (w/v) sucrose. Histological studies confirmed different developmental stages of somatic embryogenesis inHardwickia binata. Abbreviations BA N⁶-benzyladenine - Kn kinetin - NAA a-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - mMS modified Murashige and Skoog (1962) medium     

Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L.

Summary An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg/l) and indole-3-butyric acid (0.25 mg/l) induced the maximum percentage of shoot regeneration (98%) and the highest number of shoot colonies per explant (4.6) after 8 weeks of culture. Isolated shoots would elongate and proliferate when the benzyladenine concentration was lowered to 0.5 mg/l. The established protocol for shoot regeneration was employed to transform leaf disks using Agrobacterium tumefaciens carrying the plasmid pBI121. A 7.7% of the inoculated explants showed kanamycin resistance after 10 weeks of selection in a medium containing 25 mg/l of this antibiotic. The transgenic shoots obtained were rooted in the presence of 25 mg/ kanamycin and successfully acclimatized. The final percentage of transformation obtained based on beta-glucuronidase expression was 6.9%.Abbreviations BA benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog basal medium - LSD least significant difference - NOS nopaline synthase promoter - NPTII neomycin phosphotransferase (EC 2.7.1.95) - CaMV35S cauliflower mosaic virus promoter - GUS beta-glucuronidase (EC 3.2.1.31) - LB Luria Broth base - CTAB hexadecil trimethyl ammonium bromide - PCR polymerase chain reaction - X-gluc 5-bromo-4-chloro-3-indolyl-glucuronide     

Highly efficient shoot regeneration of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) using a two-stage culture procedure and assessment of genetic integrity of micropropagated plants by RAPD

A two-stage culture procedure has been developed for highly efficient shoot regeneration from leaf and internode explants of Bacopa monnieri. Adventitious shoot buds were obtained on the shoot induction medium containing Murashige and Skoog’s (MS) basal salt supplemented with 1.5 mg/l thidiazuron and 0.5 mg/l naphthalene acetic acid; these shoot buds were subcultured on the multiplication (second) medium amended with BAP (benzyl amino purine). Multiplication medium containing 0.5 mg/l BAP produced more shoots (135) and longer shoots (7.8 cm) with more nodes (6). Best response of root induction with more number of roots (16.5) and longer roots (8.7 cm) was observed in half strength MS basal medium supplemented with 1.0 mg/l IBA (indole-3-butyric acid) and 0.5 mg/l phloroglucinol. In vitro obtained plants were transferred to the field after hardening with a 100% survival rate. Random amplified polymorphic DNA analysis was carried out using five random primers. The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated plants.     

Direct shoot regeneration from foliar explants of an epiphytic orchid,Acampe praemorsa (Roxb.) Blatter and McCann

An efficient and reproducible procedure is described for the large-scale propagation of an epiphytic orchid,Acampe praemorsa (Roxb.) B latter and McCann using foliar explants. Shoot buds were induced in basal parts of foliar explants on Murashige and Skoog medium supplemented with N⁶-benzyladenine (BA), kinetin (Kn) or thidiazuron (TDZ), the latter being most effective at 1.0 mg/1. Shoots formed to a TDZ-containing medium elongated following transfer to a substrate supplemented with 2.0 mg/l 1-naphthaleneacetic acid (NAA) and 0.5 mg/1 BA. NAA at lower concentrations had no beneficial effects on shoot regeneration, whether added to the medium along with BA, Kn or TDZ. However, it promoted shoot elongation and leaf expansion. Higher concentrations of NAA suppressed shoot regeneration. The frequency of shoot regeneration was greatly influenced by the developmental stage and orientation of the leaf. Shoots regenerated from the foliar explants were rooted successfully on MS medium containing 1.0 mg/l indole-3-butyric acid. The plantlets were acclimated and eventually transferred to a garden.Abbreviations BA N⁶-Benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea)     

Highly efficient in vitro proliferation and genetic stability analysis of micropropagated Ceropegiaevansii by RAPD and ISSR markers: A critically endangered plant of Western Ghats

Ceropegiaevansii McCann (family: Asclepiadaceae), a critically endangered plant of Western Ghats has acquired significant importance due to its medicinal implications, edible tubers, and ornamental flowers. This study deals with the optimization of axillary bud proliferation using nodal explants followed by genetic stability analysis of regenerants. Maximum number of shoots (11.6 ± 1.1) was observed on the Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (4.0 mg/l) and indole-3-acetic acid (0.3 mg/l) with 85% shoot multiplication frequency. In vitro-grown shoots were rooted best in 1/2 MS medium supplemented with indole-3-butyric acid (1.0 mg/l) with an average of 10.3 ± 0.9 roots per shoot and 92% rooting frequency. Plantlets were acclimatized best (90%) in a mixture of sterile soil, sand, and coco peat (1:2:1). Micropropagated plants were subjected to random amplified polymorphic DNA and inter simple sequence repeat markers analyses. Collectively, 759 bands were generated which were monomorphic and similar to the mother plant. Findings of this study are the first report on micropropagation and assessment of genetic stability of micropropagated plantlets in C. evansii which suggests that axillary shoot proliferation can safely be used as an effective tool for propagation and conservation of C. evansii.