Effective Protocol for in Vitro Shoot Production Through Nodal Explants of Simmondsia Chinensis

Nodal explants excised from 18 to 20-year-old female plants of Simmondsia chinensis if cultured on Murashige and Skoog's medium supplemented with 20 M N⁶-benzyladenine (BA) differentiated an average of 2.7 ± 0.4 shoots in 11.5% explants. The percentage of nodal explants inducing multiple shoots enhanced significantly if in vitro raised shoots were used as source of explants. Nearly 100% cultures differentiated an average of 4.7 ± 2.0 shoots per explant on the same medium. Nearly 85% of the shoots induced roots when a pulse treatment of 50 M indole-3-butyric acid (IBA) was given prior to their transfer to semi-solid MS medium containing 10 M IBA + 0.5% activated charcoal + 1 M BA. Plantlets were gradually hardened in Soilrite and acclimatized to soil.     

Adventitious shoot production from immature embryos of white clover

Cotyledonary-stage embryos of Haifa white clover, collected 13 days after cross-pollination, were induced to form adventitious shoots primarily from the hypocotyl region. The culture medium used for the production of adventitious shoots contained 5 M thidiazuron and 0.5 M -naphthaleneacetic acid. Numerous shoot meristems were produced within the first week, discrete shoots developed by week three, small plantlets by week eight, and whole plants in soil by week ten. 95–100% of all embryos, regardless of genotype, produced adventitious shoots within four weeks with an average production of 17.5 shoots per embryo. The majority of shoots (on average 77%) were easily converted to whole plants in soil. The white clover regeneration system described is prolific, rapid and effective on a large number of genotypes.Abbreviations BA N⁶-benzylaminopurine - MS medium Murashige & Skoog medium (1962) - NAA -naphthaleneacetic acid - thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

The stimulation of cell extension by ethylene and auxin in aquatic plants

Elongation of the shoots of three aquatic plants (Hydrocharis morsus-ranae, Regnellidium diphyllum and Ranunculus sceleratus) is stimulated by treatment with ethylene or IAA. The effects of the two hormones are additive, and experiments with an ethylene biosynthesis inhibitor and silver ions indicate that the mechanisms by which ethylene and IAA stimulate growth may be different. Hydrocharis and Ranunculus leaf discs synthesize [¹⁴C]ethylene from [¹⁴C]methionine, but no [¹⁴C]ethylene is formed by Regnellidium, suggesting the existence of an alternative pathway of ethylene biosynthesis in the fern.Abbreviations IAA Indole-3-acetic acid - RBA 1,2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid     

In situ measurements of uptake of inorganic carbon and nitrogen by the aquatic liverworts Jungermannia vulcanicola Steph. and Scapania undulata (L.) Dum. in an acid stream,Kashiranashigawa, Japan

In situ uptake of inorganic carbon and nitrogen by the aquatic liverworts Jungermannia vulcanicola Steph. and Scapania undulata (L.) Dum. was measured in an acid stream, Kashiranashigawa, Japan. The uptake activities were similar in the both species. The activities were highest at the tip of shoots, and decreased gradually towards the base. Carbon uptake at the tip in the light was 10.4 × 10^–4 for J. vulcanicola and 8.1 × 10^–4 g C g dry wt^–1 h^–1 1 for S. undulata. Ammonium was effectively incorporated into the shoots, and the uptake activity at the tip was between 1.9 × 10^–5 and 5.8 × 10^–5 g N g dry wt^–1 h^–1. Nitrate uptake was smaller than ammonium uptake. The ratio of dark to light uptake in ammonium uptake experiments was larger than that in carbon uptake experiments. These results suggest that the liverworts use ammonium as a major nitrogen source, and that ammonium uptake was less dependent on light than carbon uptake.     

In vitro micropropagation of Arctostaphylos uva-ursi (L.) Sprengel.: Comparison between two methodologies

In vitro micropropagation of Arctostaphylos uva-ursi was performed to increase the number of ground cover species able to serve as substitute for members of the Rosaceae susceptible to fire blight. Explants (node segments) excised from plants growing in the greenhouse were established in vitro on a medium containing 10 M -naphthaleneacetic acid (NAA) and activated charcoal (2 g I^-1). Using in vitro grown shoots, two propagation procedures were used:- Culture of nodal fragments with 50 M NAA resulted in the growth of 6 to 7 nodes every 4 weeks, yielding 1 700 almost rootable shoots after 4 subcultures;- Development of axillary shoots obtained with media containing 25 M benzyladenine (BA) and 20 M indoleacetic acid (IAA) yielded almost 500 rootable shoots after 4 subcultures. The rate of propagation decreased after the 3^rd subculture.Percentage of in vitro rooted shoots reached 98% with diluted micronutrients and 10 M NAA but 31% of the plants died during acclimatization.Abbreviations BA benzyladenine - BM basal medium - HID high intensity discharge - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAR photosynthetic active radiation - 2iP 2-isopentenyladenine     

In vitro regeneration from hypocotyl and seedling cotyledons of tronchuda (Brassica oleracea var. tronchuda Bailey)

Four tronchuda (Brassica oleracea var. tronchuda Bailey) cultivars were tested for their ability to regenerate in vitro on Murashige & Skoog (MS) medium supplemented with 3 different combinations of -naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Explants were either axillary bud-free whole cotyledons or hypocotyls from 7-day-old darkgrown seedlings. The ability to regenerate varied by cultivars, explants and the concentration of growth regulators. Hypocotyl explants of all 4 cultivars, and cotyledon explants of 2 cultivars, developed plantlets within 4 weeks. Hypocotyl explants produced more shoots than cotyledons. Cotyledon explants produced more roots than hypocotyls. Best shoot regeneration was on MS medium supplemented with 2 mgl^-1 BAP and 0.1 mgl^-1 NAA. Portuguesa produced the most shoots. Some regenerants varied in leaf shape and phyllotaxy.Abbreviations BAP benzylaminopurine - NAA napththaleneacetic acid - IBA indolebutyric acid     

Factors affecting in vitro shoot proliferation and ex vitro establishment of mangosteen

Shoot proliferation has been achieved in Garcinia mangostana L. using seed explants. Maximum mean number of shoots per explant (16.8) was obtained from cultures on Murashige and Skoog medium supplemented with 40 mM 6- benzyladenine, and 2.5 mM -naphthaleneacetic acid and kept at 30 °C under an 8 hour photoperiod. Cultures on the same medium but supplemented with 2 g l^-1 activated charcoal produced fewer shoots. However, growth of these shoots was more organized and 75% rooting was obtained. Woody Plant Medium was not a suitable medium for shoot proliferation. Ex vitro establishment was best obtained on planting medium consisting of sand, soil and organic material (3:2:1).Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - MS Murashige & Skoog (1962) basal medium - WPM Woody Plant basal medium (Lloyd & Mc Cown 1980)     

In vitro propagation of prairie gentian

Explants of shoot tips, internodal stem sections, and leaf segments of Lisianthus, Eustoma grandiflorum (Griseb.) Schinners, Dwarf Purple were cultured in vitro on modified Murashige and Skoog (MS) media. Explants of shoot tips and internodal stem sections developed into multiple shoots, whereas, leaf segments turned chlorotic on a medium supplemented with 3 mgl^-1 benzyladenine (BA) and 0.2 mgl^-1 naphthalene acetic acid (NAA). Shoot proliferation was obtained on shoot tips and leaf segments with 3 mgl^-1 BA, but internodal stem sections became necrotic and died on this medium. Rooting was induced in cultures with multiple shoots by subculturing explants on a half-strength MS medium supplemented with 2 mgl^-1 indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to soil.     

Dark CO2-fixation and diurnal malic acid fluctuations in the submerged-aquatic Isoetes storkii

Summary In the leaves (but not corms) of the submerged aquatic Isoetes storkii malic acid concentration fluctuated from 22 eg g FW^-1 in the evening to 171 eg g FW^-1 in the morning. Associated with this was a change in titratable acidity of 152 eg g FW^-1 between morning and evening. ¹⁴C carbon was fixed in both the light and the dark, though the amount of carbon fixed in the light was more than that fixed in the dark. Autoradiographs show 88% of ¹⁴CO₂ fixed in the dark is recovered after 1 h, in malic acid and the remainder in one other unidentified product, whereas these two products contain less than 15% of the ¹⁴C fixed after 1 h exposure to ¹⁴CO₂ in the light. It is suggested that CAM metabolism in this aquatic species may be related to the low availability of CO₂ for photosynthesis during the day in its aquatic environment and that this metabolic pathway may prove common in the genus Isoetes.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Micropropagation ofPyrus andCydonia and their responses to Fe-limiting conditions

Appropriate micropropagation regimes were determined for fourPyrus species (P. amygdaliformis Vill.,P. betulaefolia Bunge,P. calleryana Dene., andP. communis L.) andCydonia oblonga L. Shoot multiplication was optimal at 10 or 20M N⁶-benzyladenine and high light intensity (135E m^–2 s^-1). Root formation of thePyrus species was stimulated by exposure of shoots to high levels (10 or 32M) of-indolebutyric acid (IBA) for 7 days or a dip in 10 mM IBA for 15 s, followed by a passage on auxin-free medium.-Naphthaleneacetic acid was more effective than IBA in stimulating rooting ofC. oblonga. The effects of Fe-limiting conditions in vitro were determined by comparing the responses of shoots and rooted plantlets to medium containing FeEDTA or FeSO₄, with or without bicarbonate. Symptoms of Fe deficiency were genotype-dependent and most severe in the presence of FeSO₄ and bicarbonate. Chlorosis was pronounced inCydonia, absent fromP. amygdaliformis andP. communis, and intermediate inP. betulaefolia andP. calleryana, indicating a good correlation between in vitro and field responses. Similar responses were obtained with rooted and unrooted shoots. These findings suggest that in vitro cultures may be used for studies of Fe-chlorosis as well as screening for tolerant rootstocks.     

Regeneration of transgenic plants from the commercial apple cultivar Royal Gala

A transformation system was developed for the commercial apple (Malus X domestica Borkh.) cultivar Royal Gala. Leaf pieces from in vitro-grown shoots were cocultivated for 2 days with Agrobacterium tumefaciens strain LBA4404 containing the binary vectors pKIWI105 or pKIWI110. Shoots were produced on a shooting medium containing kanamycin (50 mg·L^–1). A 2-day incubation period on kanamycin-free medium prior to antibiotic selection enhanced the regeneration of kanamycin-resistant shoots. The majority of the kanamycin-resistant shoots also expressed GUS (-glucuronidase) activity. The putatively transformed shoots were rooted on a medium containing kanamycin (50 mg·L^–1). Rooted plants were established in a greenhouse, and plants transformed with pKIWI110, which contains a mutant Arabidopsis acetolactate synthase gene, were shown to be resistant to the herbicide Glean. Integration of T-DNA into the apple genome was confirmed by PCR and Southern hybridization analyses.Abbreviations NAA -naphthaleneacetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine     

Agrobacterium tumefaciens-mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression

To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l^–1 -naphthaleneacetic acid (NAA) and 1.0 mg l^–1 thidiazuron (TDZ). Putative transformed shoots were selected on SR medium containing 75 g ml^–1 Km, 200 g ml^–1 Timentin, and transformation was monitored by observation of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants have set seed. GFP expression segregated in the T₁ seedlings of the two lines tested in a 3 – 1 ratio. In addition to the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern and Western blot analyses.     

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     

Micropropagation of the nickel hyperaccumulator,Hybanthus floribundus (Family Violaceae)

A protocol for micropropagation of the nickel hyperaccumulator Hybanthus floribundus (Lindley) F. Muell. (Shrub Violet) is described in this paper. Healthy callus was first produced from stem and leaf explants on a medium containing half strength Murashige and Skoog medium with 5 M N ⁶-benzylaminopurine (BA) and 0.5 M -naphthaleneacetic acid (NAA). Numerous shoots (>20 shoots per callus) were also successfully grown from callus on this medium. The exposure time of shoots to auxin was critical for successful in vitro rooting. Best rooting efficiency was obtained by transferring shoots to auxin medium (100 M indole-3-butyric acid) for 24 h and then to a medium without growth regulators (about 75% of treated shoots produced healthy roots). Importantly, cloned shoots retained their ability to hyperaccumulate nickel.     

Foliar regeneration of the endangered Red Vanda,Renanthera imschootiana Rolfe (Orchidaceae)

An in vitro method was developed to regenerate large numbers of phenotypically uniform plants from the basal parts of the leaves of flowering plants of Renanthera imschootiana Rolfe. Differentiation of up to 10 shoot buds free of callus and protocorm-like bodies occurred in 10–12 weeks from the base of a single leaf implanted in Mitra et al. (1976) medium supplemented with 2% sucrose, 2 g l^-1 peptone, 44.4 M benzyladenine (BA) and 10.7 M naphthaleneacetic acid (NAA). Subculture of the tissues in medium enriched with 10% coconut water and 35 g l^-1 ripe banana pulp resulted in the production of highest average number of 40 shoots in 12 weeks. No difference in the regeneration potential was observed among the three young leaves while mature leaves did not respond. All the leaves of the regenerated shoots were easily recultured to increase shoot multiplication. Shoots readily formed roots on transfer to a medium containing 4.4 M BA, 10.7 M NAA and 1% activated charcoal. All regenerated plants examined were normal diploids with 2n=38. Foliar meristem culture appears to have great potential for ex situ conservation and propagation of this extremely endangered orchid.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid     

In vitro plantlet regeneration of<Emphasis Type="Italic"> Schinopsis balansae</Emphasis> (Anacardiaceae)

A protocol for the in vitro regeneration of rooted plants from nodal single bud segments of 10-year-old Schinopsis balansae trees was developed. Nodal segments were harvested from actively growing shoots of plants grown from seeds and maintained in pots under greenhouse conditions, and from epicormic shoots obtained by forced flushing of branches. Culture of nodal segments on nutrient medium containing the mineral salts and vitamins of Murashige and Skoog medium at 1/4 strength (1/4 MS), supplemented with 100 mg l^–1 ascorbic acid, 3% sucrose, and 5–15 M 6-benzyladenine resulted in regeneration of multiple shoots. Rooting of regenerated shoots was observed in 1/4 MS medium with vermiculite as the substrate and supplemented with 7.5 M indolbutyric acid.     

Impact of picolinic acid on the chromium accumulation in fodder radish and komatsuna

Effects of picolinic acid (2-pyridinecarboxylic acid) and chromium(III) picolinate was studied on the chromium (Cr) accumulation of fodder radish (Raphanus sativus L. convar. oleiformis Pers., cv. Leveles olajretek) and komatsuna (Brassica campestris L. subsp. napus f. et Thoms. var. komatsuna Makino, cv. Kuromaru ) grown in a pot experiment. Control cultures, grown in an uncontaminated soil (UCS; humous sand with pH_KCl 7.48, sand texture with 12.4% clay+silt content, organic carbon 0.56%, CaCO₃ 2.2%, CEC 6.2 cmol_c kg^–1, Cr 10.6 mg kg^–1), accumulated low amounts of chromium (less than 5.4 g g^–1) in their roots or shoots. When this UCS was artificially contaminated with 100 mg kg^–1 Cr (CrCl₃) later picolinic acid treatment promoted the translocation of chromium into the shoots of both species. In fodder radish shoots Cr concentration reached 30.4 g g^–1 and in komatsuna shoots 44.5 g g^–1. Application of ethylene diamine tetra-acetic acid (EDTA) to this Cr contaminated soil had similar effect to picolinic acid. When the UCS was amended with leather factory sewage sediment (which resulted in 853 mg kg^–1 Cr in soil), Cr mobilization was observed only after repeated soil picolinic acid applications. From a galvanic mud contaminated soil (brown forest soil with pH_KCl 6.77, loamy sand texture with 26.6% clay+silt content, organic carbon 1.23%, CaCO₃ 0.7%, CEC 24.5 cmol_c kg^–1, Cd 5.0 mg kg^–1, Cr 135 mg kg^–1, and Zn 360 mg kg^–1) the rate of Cr mobilization was negligible, only a slight increase was observed in Cr concentration of fodder radish shoots after repeated picolinic acid treatments of soil. Presumably picolinic acid forms a water soluble complex (chromium(III) picolinate) with Cr in the soil, which promotes translocation of this element (and also Cu) into the shoots of plants. The rate of complex formation may be related to the binding forms and/or concentration of Cr in soil and also to soil characteristics (i.e. pH, CEC), since the rate of Cr translocation was the following: artificially contaminated soil > leather factory sewage sediment amended soil > galvanic mud contaminated soil. Four times repeated 10 mg kg^–1 chromium(III) picolinate application to UCS multiplied the transport of chromium to shoots, as compared to single 10 mg kg^–1 CrCl₃ treatment. This also suggests that chromium(III) picolinate is forming in the picolinic acid treated Cr-contaminated soils, and plants more readily accumulates and translocates organically bound Cr than ionic Cr. Picolinic acid promotes Cr translocation in soil-plant system. This could be useful in phytoextraction (phytoremediation) of Cr contaminated soils or in the production of Cr enriched foodstuffs.     

Regeneration of Picea omorika plants via organogenesis

Picea omorika plants were regenerated from embryo and seedling shoot tip cultures. Adventitious and axillary shoots were produced on 1/2 MS medium containing benzyladenine and kinetin. Benzyladenine was more effective in bud induction, whereas kinetin hastened shoot development. Excised shoots were elongated on 1/3 MS medium without growth regulators, multiplied with kinetin and rooted with or without indole-3-butyric acid.Abbreviations BA N⁶-benzyladenine - 2IP N ⁶-(²-isopenteny) adenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Cloning of Maytenus emarginata (Willd.) Ding Hou — a tree of the Indian Desert,through tissue culture

Summary An in vitro method for cloning and mass multiplication of Maytenus emarginata, a highly drought resistant tree of the Indian Desert, has been developed. Shoot segments harvested from a plus tree (30-year-old) were cultured to produce multiple shoots (10–15 shoots/explant) on MS medium containing 0.1 mgl^–1 IAA and 2.5mgl^–1 BAP. In vitro produced shoots were cut into segments and cultured on shoot proliferation medium but with only 1.0 mgl^–1 of BAP to further multiply the shoots. Isolated individual shoots were cultured on a filter paper bridge in half strength MS liquid medium containing 25 mgl^–1 of IBA for 72 h in the dark at 28±2⁰ C for induction of root(s). About 70–80 percent of shoots rooted. The treelets developed were hardened and transferred to pots. Around 20,000 plants can be obtained from a single explant within a period of 6 months. The protocol is highly reproducible and efficient.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthalene acetic acid - NOA -naphthoxy acetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - B5 Gamborg et al. (1968) medium - MS Murashige and Skoog (1962) medium