<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

Protoplasts from <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed cell line of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. regenerated to hairy roots

Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×10⁶ per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented with 2 mgl⁻¹ (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl⁻¹ (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl⁻¹ casein hydrolyzate at a plating density of 1.0×10⁵ per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators. Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast culture system would be valuable for further somatic hybridization in forage legumes.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration through somatic embryogenesis and organogenesis using cotyledons of <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) + 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D + 1 μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.     

Embryogenesis and plant regeneration from unpollinated ovary culture of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis>

Embryogenesis and plant regeneration was achieved from callus cultures derived from unpollinated ovaries of Psoralea corylifolia L. Callus was initiated from unpollinated ovaries on Murashige and Skoog (MS) medium supplemented with 2.2 μM N ⁶-benzyladenine (BA) and various concentrations of α-naphthaleneacetic acid (NAA (2.7 to 10.7 μM) or 2,4-dichlorophenoxyacetic acid (2,4-D (2.3 to 9 μM) alone or in combination. Highly organized embryogenic callus induction, embryo development, proliferation and maturation were achieved on transfer of callus clumps to MS medium supplemented with NAA (0.27 μM) or 2,4-D (0.23 μM) alone or in combination with BA (2.2 to 8.8 μM). Addition of abscisic acid (ABA) (0.95 to 5.8 μM) to the medium enhanced average numbers of cotyledonary stage embryos, the maximum number (34.6 ± 0.7) being obtained on MS medium containing 0.27 μM NAA, 2.2 μM BA and 3.8 μM ABA. Embryos germinated on MS medium supplemented with BA (0 to 8.8 μM). MS medium containing gibberellic acid (GA₃ (0.29 to 5.8 μM) enhanced embryo germination frequency, the highest frequency (66.7 %) occurring on MS medium containing 2.2 μM BA and 4.3 μM GA₃. Effect of several concentrations (3.0 to 6.0 %) of sucrose or maltose was also observed on germination of embryos. MS medium enriched with maltose supported high frequency of embryo germination.     

Optimization and mechanism of diacetyl accumulation by <Emphasis Type="Italic">Enterobacter</Emphasis><Emphasis Type="Italic">aerogenes</Emphasis> mutant UV-3

A mutant designated as UV-3 was obtained from wild-type Enterobacter aerogenes 10293 through u.v. radiation. The activities of α-acetolactate decarboxylase (Ald), lactate dehydrogenase (Ldh) and diacetyl reductase (Dr) in UV-3 were strongly attenuated, with the lowest activities at pH 7.0–7.5, and temperature between 36 and 39°C. Compared to the wild-type, the yield of diacetyl by UV-3 was increased 18.7-fold, up to 1.05 ± 0.01 g l⁻¹. Acetoin and ethanol productions were decreased by 48.4 and 71.4%, respectively, but acetate yield was increased by 34.6%. Optimum medium for diacetyl production by UV-3 contained 10% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.01% (NH₄)₂SO₄, 0.1% citric acid, 0.2% MnSO₄ and 0.2% MgSO_4, and this was determined by one-factor-at-a-time approach. Data from the five level central composite designs demonstrated that initial pH of 7.0, temperature of 37°C and rotational speed of 180 rev/min were optimum processing parameters for diacetyl production. The maximum yield of diacetyl could reach 1.35 g l⁻¹ in a 5-l bioreactor. These results showed an enhancement of the non-enzymatic oxidative decarboxylation of α-acetolactate and a decrease in the activities of Ald, Ldh and Dr as a consequence of diacetyl accumulation in UV-3.     

<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.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

Plant regeneration from leaf protoplasts of <Emphasis Type="Italic">Solanum virginianum</Emphasis> L. (<Emphasis Type="Italic">Solanaceae</Emphasis>)

Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 10⁶/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS) medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid. Regenerated plants have normal morphology.     

In vitro regeneration in <Emphasis Type="Italic">Dierama erectum</Emphasis> Hilliard

The genus Dierama comprises plants with a potential to be developed as ornamentals. D. erectum seeds were decontaminated and germinated on 1/10th strength Murashige and Skoog (Physiol Plant 15:473–497, 1962) (MS) media without plant growth regulators or sucrose. In an experiment investigating the effects of 6-benzyladenine (BA), meta-Topolin (mT), kinetin (KIN) and zeatin (Z) with or without α-naphthaleneacetic acid (NAA), the highest shoot number per hypocotyl (4.20 ± 0.51) was obtained from MS medium supplemented with 1.0 μM Z after 8 weeks. This was followed by a combination of 2.0 μM KIN and 2.0 μM NAA with 3.67 ± 0.81 shoots per explant. BA treatments produced 3.20 ± 0.22 shoots per hypocotyl explant when 2.0 μM was combined with 1.0 μM NAA, while mT gave 3.09 ± 0.99 shoots per explant when 2.0 μM mT was combined with 2.0 μM NAA. Adventitious shoot regeneration was optimised when shoots were grown under a 16-h photoperiod at 100 μmol m⁻² s⁻¹ on MS medium supplemented with 1.0 μM BA. This resulted in an average of 12.73 ± 1.03 shoots per hypocotyl explant. Various concentrations of ancymidol, activated charcoal and sucrose did not promote in vitro corm formation of this species. Plants rooted successfully after 8 weeks on MS medium supplemented with 1.0 μM indole-3-butyric acid (IBA) and had an average root number of 2.73 ± 0.40. After 2 months of acclimatisation, plants had formed corms. The largest corms (of diameter 0.45 ± 0.03 cm) were produced in plants pre-treated with 0.5 μM IBA. The highest plant survival percentage of 73% was also associated with this treatment.     

<Emphasis Type="Italic">In vitro</Emphasis> clonal propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> (L.) wall. through nodal explants from mature trees

Summary In vitro clonal propagation of 18–20-yr-old Holarrhena antidysenterica tress has been achieved by employing nodal explants. The tree explants showed marked seasonal variation in their morphogenic response under in vitro conditions. Maximum response was obtained from the beginning of May to the end of July, followed by a gradual decline, finally dropping to zero from October to February. The explants induced multiple shoots only on cytokinin-containing medium. Several cytokinins [N⁶-benzyladenine (BA), N⁶-(2-isopentenyl) adenine (2ip), 6-furfuryl aminopurine (Kn), and adenine sulfate (Ads)] were assayed. The best response was achieved with 15 μM BA in which 62.5% of cultures produced 2.75±0.2 shoots per explant with 3.56±0.2 cm average length. Amongsth the three heavy metals assayed, silver nitrate (AgNO₃) significantly improved the response. This compound enhanced both the percentage of responding cultures (86.6%) and the average shoot number (4.73±0.2) at a concentration of 20mgl⁻¹. Further improvement in the morphogenic response occurred when explants from in vitro shoots were employed instead of mature trees. In this case, the percentage of morphogenic cultures was increased to 100% at the third subculture with an average of 11.45±0.3 shoots per explant. Regenerated shoots were rooted in half-strength Murashige and Skoog medium with 10 μM indole-3-acetic acid. The plantlets were successfully acclimatized in soil.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

Micropropagation of <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis>

An efficient protocol for micropropagation of Harpagophytum procumbens DC., an endangered African medicinal plant, was developed. Maximum shoot multiplication without callus was obtained from nodal explants cultured on Murashige and Skoog (MS) basal salts plus Gamborg’s (B₅) vitamins supplemented with 0.1 mg dm⁻³ indole-3-acetic acid and 5.0 mg dm⁻³ kinetin. The shoots were subsequently subcultured every 3 weeks on the same medium. Detached axillary shoots were transferred to MS basal salts plus B₅ vitamins supplemented with various concentrations of α-naphthalene-acetic acid or indole-3-butyric acid (IBA), ranging from 0.5 to 2.5 mg dm⁻³ and 100 % rooting and optimal subsequent acclimatization was achieved on 1.0 mg dm⁻³ IBA. After 4 weeks of culture, the rooted shoots (>5 cm) were planted in pots containing peat, vermiculite and bark (2:1:1), covered with plastic domes and maintained at 25 °C for 2 weeks before being transferred to a glasshouse. Plant survival was about 40 %.     

<Emphasis Type="Italic">In vitro</Emphasis> multiplication of heavy metals hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

A micropropagation protocol through multiple shoot formation was developed for Thlaspi caerulescens L., one of the most important heavy metals hyperaccumulator plants. In vitro seed-derived young seedlings were used for the initiation of multiple shoots on Murashige and Skoog (MS) medium with combinations of benzylaminopurine (BA; 0.5–1.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0–0.2 mg dm⁻³), gibberellic acid (GA₃; 0–1.0 mg dm⁻³) and riboflavin (0–3.0 mg dm⁻³). The maximum number of shoots was developed on medium containing 1.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA. GA₃ (0.5 mg dm⁻³) in combination with BA significantly increased shoot length. In view of shoot numbers, shoot length and further rooting rate, the best combination was 1.0 mg dm⁻³ BA + 0.5 mg dm⁻³ GA₃ + 1.0 mg dm⁻³ riboflavin. Well-developed shoots (35–50 mm) were successfully rooted at approximately 95 % on MS medium containing 20 g dm⁻³ sucrose, 8 g dm⁻³ agar and 1.0 mg dm⁻³ indolebutyric acid. Almost all in vitro plantlets survived when transferred to pots.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Capsicum chinense</Emphasis> Jacq.

An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA). Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction. A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully established in the soil.     

A simple cryopreservation protocol of <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L. embryogenic calli by encapsulation-vitrification

Embryogenic calli of Dioscorea bulbifera L. were successfully cryopreserved using an encapsulation-vitrification method. Embryogenic calli were cooled at 6°C for 5 days on solid MS medium (Murashige and Skoog 1962) containing 2 mg L⁻¹ Kinetin (Kn), 0.5 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.5 mg L⁻¹ 2,4-dichlorophenoxy-acetic acid (2,4-D). These were prior precultured on liquid basal MS medium enriched with 0.75 M sucrose at 25 ± 1°C for 7 days. Embryogenic calli were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dropped in a 0.1 M CaCl₂ solution containing 0.4 M sucrose at 25 ± 1°C. After 15 min of polymerization, Ca-alginate beads (about 4 mm in diameter) were dehydrated for 150 min at 0°C in a PVS₂ solution [30% glycerol, 15% ethylene glycol, and 15% dimethyl sulfoxide (w/v)] containing 0.5 M sucrose. The encapsulated embryogenic calli were then plunged directly into LN (liquid nitrogen) for 1 h. After rapid thawing in a water bath (37°C; 2 min), the beads were washed 3 times at 10-min intervals in liquid basal MS medium containing 1.2 M sucrose. Following thawing, the embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, 0.09 M sucrose and 0.75% (w/v) agar (embryoid induction medium) and cultured under light conditions of 12-h photoperiod with a light intensity of 36 μmol m⁻² s⁻¹ provided by white cool fluorescent tubes after a 2-day dark period at 25 ± 1°C. After 30 days, the embryoids developed from embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, NAA 0.5 mg L⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar (regeneration medium). After 60 days, the embryogenic calli developed normal shoots and roots. No morphological abnormalities were observed after plating on the regeneration medium. The survival rate of encapsulated vitrified embryogenic callus reached over 70%. This encapsulation-vitrification method appears promising as a routine and simple method for the cryopreservation of Dioscorea bulbifera embryogenic callus.     

Somatic embryogenesis and regeneration of <Emphasis Type="Italic">Vigna radiata</Emphasis>

An efficient regeneration protocol via somatic embryogenesis was optimized for mung bean [Vigna radiata (L.) Wilczek; cv. Vamban 1]. Primary leaf explants were used for embryogenic callus induction in MMS medium (Murashige and Skoog salts with B5 vitamins) containing 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D), 150 mg dm⁻³ glutamine and 3 % sucrose. Fast growing, highly embryogenic cell suspensions were established from 21-d-old calli in MMS medium supplemented with 0.5 mg dm⁻³ 2,4-D and 50 mg dm⁻³ proline (Pro), and maximum recovery of globular (39.0 %), heart-shaped (26.3 %) and torpedo-stage (21.0 %) somatic embryos were observed in this medium. Mature cotyledonary-stage somatic embryos were cultured for 5 d in half strength B5 liquid medium containing 0.05 mg dm⁻³ 2,4-D, 20 mg dm⁻³ Pro, 5 μM abscisic acid, 1000 mg dm⁻³ KNO₃, 50 mg dm⁻³ polyethylene glycol (PEG 6000) and 30 g dm⁻³ D-mannitol. Mature somatic embryos were germinated after dessication for 3 d and complete development of plantlets accomplished in MMS medium containing 30 g dm⁻³ maltose, 0.5 mg dm⁻³ benzyladenine and 500 mg dm⁻³ KNO₃. Profuse lateral roots, and regeneration frequency (up to 60 %) were observed in half-strength MMS medium containing 0.5 mg dm⁻³ indolebutyric acid (IBA). The regenerated plants were grown to fruiting and were morphologically normal and fertile.     

Foliar nutrition of <Emphasis Type="Italic">in vitro</Emphasis>-cultured <Emphasis Type="Italic">Prosopis chilensis</Emphasis> (Molina) Stuntz shoots

Summary Foliar nutrition has been conceived as a possible means of overcoming the recalcitrance of Prosopis chilensis (Molina) Stuntz explants to standard in vitro culture. The foliar uptake of cations (K from 20 gl⁻¹ KNO₃ and Ca from 50 gl⁻¹ CaCl₂), anions (NO₃ from 50 gl⁻¹ KNO₃ and PO₄ from 50 gl⁻¹ NaH₂PO₄), and glucose from a 100 mg l⁻¹ solution studied. All of the nutrients examined were absorbed. The efficacy of foliar nutrition in prolonging the vigor of micropropagated P. chilensis shoot tips was compared with nutrients supplied as a liquid to the base of the stem (liquid) or as an agar-solidified medium (agar). A foliar-feeding apparatus was constructed that employed pressurization of the medium reservoir to drive the medium into the culture vessel with a passive return by a siphoning effect. The medium used was Murashige and Skoog with 30 gl⁻¹ sucrose, 0.1 mgl⁻¹ benzylaminopurine, and 1 mgl⁻¹ indole-3-butyric acid. Over a 9-wk test period it was found that explants cultured by foliar nutrition performed significantly better than those grown on agar for shoot length, nodal production, and leaf retention; and better than liquid MS for node production. There was no significant difference among the three treatments in percentage survival, percentage rooting, or the mean number of roots.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Eucalyptus phylacis</Emphasis> L. Johnson and K. Hill., A critically endangered relict from Western Australia

Summary In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal, which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m⁻²s⁻¹) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with 0.5 μM zeatin and 1 μM gibberellic acid, A₄ isomer (GA₄). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5 μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA₄ for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture.