A tissue culture method for rapid clonal propagation of mature trees of Eucalyptus torelliana and Eucalyptus camaldulensis

Multiple shoots were induced from nodal segments of mature Eucalyptus torelliana F. Muell. and E. camaldulensis Dehnh trees on Murashige and Skoog's medium supplemented with Kn, BAP, Cal.Pan and Bio. Incubation in semi-solid media at 15°C with continuous illumination followed by growth in agitated liquid media was essential for shoot induction and development in primary explants of E. camaldulensis. For culture of E. gorelliana, growth in agitated liquid media alone was sufficient. Rooting could be induced in shoot cultures of E. torelliana by treatment with NAA whereas treatment with a mixture of IAA, IBA, IPA and NAA was essential for E. camaldulensis. After auxin treatment, transfer to a charcoal-containing medium was necessary. Rooted plantlets could be successfully transferred to pots and field. By this method it is estimated that about 50,000 plantlets of E. torelliana and 20,000 of E. camaldulensis can be produced, in a year, from a single nodal segment of a mature tree.Abbreviations Kn Kinetin - BAP 6-Benzylaminopurine - Ca.Pan Calcium Pantothenate - Bio Biotin - NAA -Naphthalene acetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid Communication No. 3315     

Tissue,cell culture and micropropagation of Mandevilla velutina,a natural source of a bradykinin antagonist

Leaf, stem and root explants of Mandevilla velutina were cultured in vitro and produced vigorous callus in LS basal medium containing one auxin (2,4-D or NAA) plus BAP. Calli can be subcultured indefinitely with vigorous growth. Subculture of calli to NAA (1.0 mg/l) plus BAP (5.0 mg/l) caused profuse regeneration of shoots. Isolated shoots were rooted in basal medium plus NAA (5.0 mg/l) or IBA (8.0 mg/l). Rapidly growing cell suspensions can be easily obtained from friable callus cultured in liquid medium.Abbreviations LS Linsmaier & Skoog - 2,4-D 2,4 dichlorophenoxi-acetic acid - NAA -naphthalene-acetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid     

Plant regeneration from callus cultures of Valeriana wallichii DC.

Petiole expiants of Valeriana wallichii. DC., a threatened medicinal plant, were used for inducing callus. Optimum callus formation was observed on Murashige and Skoogs' (1962) medium supplemented with 3.0 mg/l NAA and 0.25 mg/l Kn. Shoot regeneration was achieved upon transferring the callus to medium containing 1.0 mg/l Kn and 0.25 mg/l NAA. Complete plantlets were obtained on the same medium or upon transfer of the regenerated shoot buds to medium containing 5.0 mg/l Kn and 1.0 mg/l IAA. Nearly a thousand callus regenerated plants were successfully transferred to the field following previously standardized hardening procedures.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichloro phenoxyaceticacid - 2iP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's medium (1962) - NAA -napthalene aceticacid - Z Zeatin     

Regeneration of plantlets from cell suspension culture derived callus of white poplar (Populus alba L.)

Friable calli derived from the stem tissues of Populus alba were used to establish cell suspension cultures which were characterized for in vitro growth and regeneration capacity. Suspended cells and callus recovered from these cells were maximal on a fresh weight basis using MS liquid medium containing 0.44 M BAP and 4.52 M 2,4-D. Shoot regeneration from the recovered callus was observed within 30 to 40 days of culture. The number of shoots was increased by subculturing the shoot-forming callus 2 to 3 times on MS medium supplemented with 19.7 M 2iP and 0.05 M IBA. Regenerated shoots were easily rooted on half-strength MS medium lacking growth regulators, and the plantlets were transferred to pots containing vermiculite for greenhouse growth.Abbreviations BAP 6-benzylaminopurine - 2iP 2-isopentenyladenine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS medium Murashige and Skoog medium (1962)     

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     

Regeneration of Medicago truncatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants

Plant regeneration has been achieved by somatic embryogenesis in Medicago truncatula Gaertn. (barrel medic) c.v. Jemalong, an annual legume species. Regenerated plants were obtained from cultured leaf tissue explants on a four-step modified B5 basal medium. Induction of embryo formation occurred on a medium containing 10 M NAA and 10 M BAP, and embryo maturation was promoted after transfer to a medium containing 1 M NAA and 10 M BAP. Shoot development, secondary somatic embryogenesis and occasional plantlet development occurred on a subsequent transfer to 0.1 M NAA and 1 M BAP. Plantlet formation could also be completed by transfer of well developed shoots to 0.05 M NAA. A high frequency of primary somatic embryos could only be obtained by using the same culture protocol with tissue from regenerated plants. Explants from regenerated plants showed a large increase in the number of primary embryos per callus and the number of calli producing embryos. Explants from plants derived from the seed of one regenerated plant also showed increased embryo formation. Although high embryo formation rates can be reproducibly obtained from this seed, embryo conversion rates to plants are currently low.Abbreviations BAP 6-benzylaminopurine - B5 medium of Gamborg et al. 1968 - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium of Murashige and Skoog 1962 - NAA 1-naphthaleneacetic acid     

In vitro propagation of guayule (Parthenium argentatum) — a rubber yielding shrub

Nodal explants (0.5 to 0.8 cm long) isolated from 2-year old shrubs of guayule, Parthenium argentatum Gray, when cultured on MS medium supplemented with different concentrations of KN, BAP, 2,4-D, 2,4-D + BAP, NAA and NAA + BAP produced callus tissues and shoots simultaneously with varying frequencies. Shoots were regenerated with a high frequency (80–88%) from callus on MS medium containing NAA + BAP with or without glutamine. Addition of glutamine to these media improved considerably the number of shoots formed from a known amount of callus. Shoots could be regenerated from 200 day old callus cultures with a very high frequency but the organogenetic capacity declined thereafter. Increase in the concentration of sucrose (upto 4%) significantly enhanced the shoot forming ability of callus, but higher concentrations (6%) suppressed it. Rooting was induced only in dark when IAA, IBA and NAA were used, but 2,4-D could induce them both in light and dark. The system is suitable for the mass propagation of this important rubber yielding plant.Abbreviations MS Murashige and Skoog (1962) - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - KN Kinetin - BAP 6-Benzylaminopurine     

Micropropagation of Uniola paniculata L.

Uniola paniculata L. is a major sand-dune stabilizing grass which is being utilized to prevent shoreline erosion. In vitro cultured caryopses of U. paniculata produced callus on MS medium supplemented with 22.5 M 2,4-D, 4.4 M BA and 87.6 mM sucrose. Shoot induction occurred after these calli were inoculated onto the same medium without 2,4-D. Rooting of in vitro-derived shoots occurred when transferred to a one-half strength MS medium containing 43.8 mM sucrose and 14.7 M IBA. Plantlets were planted after the roots reached a length greater than 20 mm.Abbreviations BA N-(phenyl-methyl)-lH-purine-6-amine - IBA lH-indole-3-butanoic acid - MS Murashige and Skoog (1962) - 2,4-D (2,4-dichlorophenoxy) acetic acid - TC agar tissue culture agar, (K.C. Biologicals, Lenexa, KS) - Subdue methaxyl:N-(2,6-dimethylphenyl)-N-(methyoxyacetyl) alanine methyl ester (CIBA- GEIGY)     

Development of in vitro procedures for regeneration of petiole and leaf expiants and production of protoplast-derived callus in Tanacetum vulgare L. (Tansy)

Tanacetum vulgare (Tansy) was established in vitro on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) using shoot tips and embryos. From petiole expiants 93% formed callus, and 27% produced shoots on MS medium containing 4.5 mg l^-1 NAA and BAP. NAA alone induced root formation from leaf expiants. Up to 7 ×10⁶ viable protoplasts were obtained by macerating 1 g of leaves in 0.5 % Macerozyme R-10, 1.0% Cellulase R10, and 1.0% Cellulysin. Cell division was observed 3–4 days after protoplast isolation at the optimum plating density of 0.2-0.4×10⁶ cells ml^-1. A total of 350 protoplast-derived calluses were produced on which nodules with meristematic zones developed. Roots regenerated on MS medium supplemented with BAP 3.0 mg 1^-1, NAA 2.0 mg l^-1, and 250 mg l^-1 casein hydrolysate, however no shoots have been obtained yet.Abbreviations BAP 6-benzylaminopurine - CH casein enzymatic hydrolysate - 2.4 D dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellic acid - IBA indole butyric acid - IPA 6-dimethylallylamino purine - KIN Kinetin - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid     

In vitro propagation of Eucalyptus grandis L. by tissue culture

Multiple shoots were induced from nodal segments of five year old trees of Eucalyptus grandis L. on solid medium containing Murashige and Skoog's (MS) Basal medium supplemented with additional thiamine, BAP and NAA. Rooting could be achieved from shoot culture on half strength MS salts or white's medium supplemented with low auxins like IAA, IBA and NAA.Abbreviations Kn Kinetin - BA 6-Benzyl adenine - 2iP Isopentyl adenine - NAA -Naphthalene acetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - GA Gibberellic acid - PVP Polyvinyl pyrrolidone     

Effect of acetone in the culture medium on the regeneration efficiency and on the resistance to root rot of regenerants of various plant species

Various expiants of wheat, onion, garlic, and cabbage were examined for their regeneration efficiency in the presence of acetone in the culture medium. Acetone at concentrations between 1.5% and 3.0% (w/v) was shown to increase the degree of callusing and meristem formation, as well as the capacity to regenerate. It also significantly increased the survival and regeneration efficiency of expiants inoculated with Agrobacterium tumefaciens, a bacterium known to reduce expiant viability. Resistance to root rot disease, caused by Helminthosporium sativum, was also increased. Thus acetone in the culture medium increases regeneration efficiency on selective media and under other stressful growth conditions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthalene acetic acid - BAP 6-benzylaminopurine - IBA indolebutyric acid     

Plantlet regeneration from mature zygotic embryos of eastern white pine (Pinus strobus L.)

Plants were regenerated from whole embryo explants obtained from eastern white pine (Pinus strobus L.) seeds. Embryos were surgically removed and axenically cultured to induce buds in vitro on a modified Murashige and Skoog medium containing various concentrations of 6-benzylaminopurine. Embryos remained on bud induction medium for 21 days and then were transferred to the same basal medium without 6-benzylaminopurine to promote bud development and subsequent shoot elongation. The medium containing 10 M 6-benzylaminopurine induced the greatest number of shoots per embryo. Rooting was achieved by direct transfer of the shoots to a non-sterile artificial soil mixture followed by multiple treatments with 15 nM 1-naphthaleneacetic acid. Regenerated seedlings are currently growing under greenhouse conditions.Abbreviations NAA 1-napthaleneacetic acid - BA 6-benzylaminopurine - SIM shoot induction medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - TIBA Triiodobenzoic acid - 2iP 2-isopentenyl adenine     

Rapid micropropagation for Mucuna pruriens f. pruriens L.

A rapid micropropagation system was developed for Mucuna pruriens f. pruriens using explants from 1-week-old aseptically grown seedlings. Multiple shoot regeneration occurred following an initial callus growth on Revised Tobacco (RT) medium supplemented with 2.7 M NAA and 9.8 M 2iP. Maximum number of shoot regeneration was achieved only from seedling explant 6 to 7 days old. More than 90% of the regenerated shoots could be rooted on half-strength liquid RT medium supplemented with 2.7 M NAA. Plantlets readily adopted to greenhouse conditions. This system provides a new tool for micropropagation of Mucuna pruriens f. pruriens, an important medicinal plant.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - 2iP 2-isopentenyladenine - AdS adenine sulphate - NAA alpha-naphthalene acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - RT Revised Tobacco medium (Kaul and Staba, 1968)     

Induction of division and differentiation of somatic embryos in the leaf epidermis of Gaillardia picta

Somatic embroys and subsequent plant regeneration were obtained from isolated leaf epidermis of Gaillardia picta. Abaxial and adaxial epidermal peels (monolayer) from 45 days old aseptic seedlings were isolated and segments measuring 5 mm x 3 mm were cultured on B₅ basal medium supplemented with various growth regulators such as naphthaleneacetic acid or indolebutyric acid and benzylaminopurine or kinetin. Within 12 h of culture the epidermal cells showed receding of cytoplasm from the walls. After 48 h of incubation 3 or 4 localized zones, each consisting of 3–8 cells that accumulated cytoplasm and stained densely, were observed. Mitotic divisions occurred in these zones on day 3 of culture and localized masses of callus were observed in 95% of the cultures after 10 days. In another 5 days, the callus differentiated somatic embryos or roots, depending on the growth regulators and their concentration in the medium.Abbreviations Used BAP 6-benzylamiopurine - IBA indolebutyric acid - Kn kinetin - NAA -naphthaleneacetic acid     

Studies on endosperm culture of Annona squamosa Linn

Mature endosperm tissue excised from germinated seeds (2–4 days after radicle emergence) of Annona squamosa grew and proliferated on White's basal medium supplemented with two cytokinins, an auxin and gibberellic acid. The callus obtained could be periodically subcultured. Shoot differentiation and root induction were obtained from callus on media of different compositions. Analyses of the root and young leaf tips showed triploid number of chromosomes (3n=21).Abbreviations Kin Kinetin - BAP 6-Benzylaminopurine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA naphthalene abetic acid - GA₃ Gibberellic acid Communication No. 3885     

Plant regeneration and ploidy variation in culture derived plants of Asclepias curassavica L

Clonal propagation of medicinal milkweed, Asclepias curassavica L. (Asclepiadaceae) was achieved by culturing excised nodes on MS medium (Murashige and Skoog, 1962) supplemented with different hormone combinations. Both BAP and Kn were found equally effective for shoot initiation. IAA and NAA were found suitable for root induction. Combinations of Kn and NAA induced both roots and shoots after 30 days of culture. Chromosomal variation was observed in the roots of in vitro regenerated plants. Regenerants with higher chromosome number (33; 2n=22) obtained on MS medium in response to 9.2 M Kn+10.7 M NAA showed vigorous growth and higher propagation rates in culture than the plants possessing less than the diploid chromosome number (2n–2=20, 2n–4=18). Such variations are more likely due to genetic fitness of different aneuploids grown on a particular nutrient medium.Abbreviations IAA 3-Indoleacetic acid - NAA Naphaleneacetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indolebutyric acid - Kn Kinetin - BAP Benzyladenine - GA₃ Gibberellic acid - S Shoot - R Root     

In vitro propagation of Glehnia littoralis from shoot-tips

Shoot cultures of Glehnia littoralis F. Schmidt ex Miq. (Umbelliferae) were established by placing shoot tip explants on Linsmaier and Skoog medium with 1 M NAA and 10 M BAP. Shoots were multiplied on the basal medium supplemented with 0.3 M NAA and 3 M BAP and rooted on medium containing either 1 M IBA or 3–10 M IAA. Plantlets survived in pots without any covering. This unique characteristic of the plantlets was ascribed partly to a well-developed cuticle on the surface of the leaf and the small ratio of surface area to fresh weight of a leaf blade in comparison with those of other species whose plantlets needed coverings after potting. The regenerated plantlets were finally transferred to soil.Abbreviations IAA potassium indole-3-acetate - IBA indole-3-butyric acid - IPA indole-3-propionic acid - NAA potassium 1-naphthaleneacetate - 2,4-D sodium 2,4-dichlorophenoxyacetate - BAP 6-benzylaminopurine - 2-iP N⁶-(2-isopentenyl)adenine     

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing -naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Micropropagation of Phytolacca dodecandra through shoot-tip and nodal cultures

A procedure for micropropagation of endod (Phytolacca dodecandra) is described. BA at 0.44 M produced 3.1 new shoots per expiant in six weeks using shoot tips. Nodal expiants, however, produced up to 4.7 shoots per explant on medium with 0.44 M BA and 0.27 M GA,. IBA at 0.49 M induced 90% rooting with minimal callus. Plantlets were successfully transferred to the greenhouse and some staminate clones produced flowers after six months.Abbreviations BA 6 benzylaminopurine - kinetin 6-furfurylaminopurine - 2iP N⁶-(2-isopentyl)adenine - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - GA₃ Gibberellic acid - IBA indole-3-butyric acid     

The effect of auxin type and concentration on pecan (Carya illinoinensis) somatic embryo morphology and subsequent conversion into plants

Summary Embryogenic cultures were initiated from immature pecan zygotic embryos. Explants were induced for one week on Woody Plant Medium with either -naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid at 2, 6 or 12 mg/l, then subcultured monthly to fresh basal medium. Observations were made on callus production, embryo formation, and embryo morphology. Somatic embryo morphology and overall callus proliferation were affected by auxin type. Callus proliferation was less extensive and more somatic embryos resembling zygotic embryos were obtained from cultures initiated with -naphthaleneacetic acid than with 2,4-dichlorophenoxyacetic acid. Repetitive somatic embryogenesis was obtained in all auxin treatments. Conversion into plantlets was affected by somatic embryo morphology in that embryos with poorly developed apices exhibited lower percentages of conversion than those with well developed single or multiple apices. Consequently, although more embryos were obtained with 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid was the superior auxin for production of somatic embryos more likely to convert into plants.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - WPM Woody Plant Medium (Lloyd & McCown 1980)