Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

Sur la biologie deMicrophthalma europaea Egg., tachninaire parasite des larves deScarabaeidae

Zusammenfassung Die Laborzucht aufeinanderfolgender Generationen vonMicrophthalma europaea egg. hat es uns m?glich gemacht, die Entwicklung dieser Tachinide bei 25°C festzustellen. Die Dauer der Stadien ist 3–4 Tage für das erste, 4–7 Tage für das zweite und 8–12 Tage für das dritte Larvenstadium, 14 Tage für die Nymphe, 14–18 Tage für die Reifungsperiode der Weibchen. Die mittlere Lebensdauer für die weiblichen Fliegen ist 25 Tage und 20 Tage für die m?nnlichen. Auch über den Einfluss der Umwelt hat man Erfahrungen gesammelt und zwar über den Einfluss der Temperatur und der Feuchtigkeit auf die Fruchtbarkeit dieser Zweiflügler, die selten h?her ist als 300–400 Planidia pro Weibchen und über die M?glichkeiten im Boden zu überleben, ehe sie in die Wirtslarve eindringen. Unter den Umst?nden dieses Versuches ist die Entwicklung nur in den Larven vonCetoniinae und vonDynastinae m?glich; die Larven vonMelolonthinae werden nicht, oder sher wenig parasitiert. Unter den empfindlichen Arten variiert die Menge der angegriffenen Individuen mit der Art ebenso wie die Anzahl und das Gewicht der pro Wirtslarve geformten Puppen. Die Larven vonPotosia cuprea F. und vonPhyllognathus silenus F. sind güstiger für die Entwicklung als z.B. die Larven vonOryctes nasicornis L. oder vonOxythyrea funesta Poda. Die Vervollkommnung der Zuchttechnik erlaubt es, die für einen biologischen Bek?mpfungsversuch gegen Scarabeiden-Larven n?tige Anzahl Parasiten zu produzieren.

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Cette étude préliminaire a été effectuée en liaison avec.E. Biliotti, Directeur de la Station de Lutte biologique et de Zoologie agricole d'Antibes, spécialiste en la matière. Nous l'en remercions vivement.     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

Studies on the Formation of Roots and Shoots in Wheat Callus Cultures

Callus tissues were initiated from root, embryo and inflorescenceexplants of wheat. These callus cultures were used to studythe formation of roots and shoots in the absence and presenceof selected plant hormones. On a basal medium alone, only newly-initiatedembryo callus formed both roots and shoots while root callusonly formed roots. Inflorescence callus showed no signs of differentiation.The regenerative capacity of root and embryo callus tissueson medium lacking hormones decreased with increasing periodsof culture. Calluses which failed to differentiate in the absenceof hormones were selected for studies on hormone-mediated differentiation.NAA (1 mg 1^–1) was effective in inducing roots from allcalluses irrespective of their origin or age. In contrast, shootformation was elicited by incubating newly-formed callus onbasal medium supplemented with kinetin (5 mg 1^–1) andNAA (1 mg 1^–1) but rapidly decreased with longer periodsof culture. The differences observed in differentiation of thecallus in the absence and presence of hormones is discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and plant establishment of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. F.) Merrill: Petiole callus culture

Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age.     

In vitro propagation of Clianthus formosus (Sturt's desert pea) an Australian native legume

Hypocotyl and cotyledon segments of Clianthus formosus were cultured on a modified deFossard medium M supplemented with cytokinins. 62% of cultures on medium with 20 μM BA produced callus which subsequently gave rise to shoots. 40% of shoots excised from callus produced roots after transfer to auxin-rich media (20 μM NAA or 10 μM IBA+10 μM NAA). Root production was enhanced following a 7-day dark treatment. 32% of nodes from mature plants produced multiple shoots on 2 μM BA+2 μM KIN. 30% of these shoots rooted on medium without hormones. 70% of rooted plantlets were successfully transferred to potting medium and glasshouse conditions. A period of cold treatment (10 days at 5°C) reduced vitrification from 68 to 22% of cultures.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

High-frequency plant regeneration from cotyledon callus of <Emphasis Type="Italic">Acacia sinuata</Emphasis> (Lour.) Merr

Summary A new reliable protocol for the induction of adventitious shoots and plant regenertion from cotyledon-derived callus of Acacia sinuata has been developed. Calluses were induced from cotyledon explants on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar or 0.15% phytagel, 8.1 μM α-naphthaleneacetic acid, and 2.2 μM 6-benzylaminopurine (BA). High-frequency regeneration of adventitious buds from callus was achieved when cultured on half-strength MS medium supplemented with 10% coconut water, 13.3 μM BA, and 2.5 μM zeatin. Histological studies revealed that the regenerated shoots originated from the callus. Among the various carbohydrates tested, sucrose at 87.6 mM was optimum for shoot-bud induction. Addition of 1.7 μM gibberellic acid along with 4.4 μM favored shoot elongation. In vitro-raised shoots produced prominent roots when transferred to half-strength MS medium supplemented with 7.4 μM indole-3-butyric acid. Rooted plants, thus developed, were hardened and successfully established in soil (45%). This protocol yielded an average of 40 plantlets per cotyledon explant over a period of 3 mo.     

Plant regeneration from protocorm-derived callus of <Emphasis Type="Italic">Cypripedium formosanum</Emphasis>

Summary Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the medium with 4.44 μM N⁶-benzyladenine after 8 wk of culture. The regenerated protocorm-like bodies formed shoots and roots on medium containing 1 g l⁻¹ activated charcoal and 20 g l⁻¹ potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established.     

Rapid mass propagation of Tylophora indica Merrill via leaf callus culture

An efficient protocol has been developed for rapid mass propagation of Tylophora indica from leaf derived callus. Optimal callus was developed from leaf explants on Murashige and Skoog (MS) basal medium supplemented with 10 2,4,5-T. Adventitious shoots were regenerated (85%) from the surface of the callus on MS medium supplemented with 5 M Kinetin. Individual elongated shoots were rooted on half-strength MS medium containing 0.5 M IBA. Regenerated plantlets with well developed shoots and roots were successfully transferred to soil. The study demonstrated a dedifferentiated callogenic propagation route via adventitious shoot development in T. indica, which could be useful for large scale multiplication of this endangered medicinal plant.