In vitro cultivation of donor quince shoots affects subsequent morphogenesis in leaf explants

The effect of in vitro cultivation of donor shoots on subsequent morphogenesis in leaf explants of quince (Cydonia oblonga Mill.) clone BA29 was investigated. Proliferating donor shoots were cultured in ventilated or closed vessels under different photosynthetic photon flux densities (PPFD; 200 and 100 μmol m⁻² s⁻¹) with 0, 15, 30 g dm⁻³ sucrose. Shoots grown in ventilated vessels, especially with sucrose at 15 or 30 g dm⁻³, were better developed with fully expanded leaves compared to those in standard closed vessels. Leaves collected from pre-treated donor shoots were used to assess regeneration capacity. Somatic embryo production was highest in leaves harvested from shoots cultured in closed vessels with 30 g dm⁻³ sucrose and in ventilated vessels with 15 and 30 g dm⁻³ sucrose and under high PPFD which was, in comparison with the control treatment (closed vessel, 30 g dm⁻³ sucrose and low PPFD), about 2 to 2.5 times higher. A similar response was observed for root regeneration.     

In vitro cormlet development in Crocus sativus

An improved protocol for generation of viable cormlets from tissue culture derived shoots of saffron has been developed. Multiple shoots were generated from apical buds, small corms and in vitro developed single shoots. Bunches of two to three shoots when cultured on half strength Murashige and Skoog (MS) medium containing 3 mg dm⁻³ benzyladenine (BA) and 80 g dm⁻³ sucrose developed 1.89 cormlets per shoot bunch with an average fresh mass of 1.18 g. It took nine months from culture of apical buds to the harvest of cormlets but under field conditions 22 months. Sucrose appeared to be essential for cormlet induction as no cormlets were developed in the medium devoid of sucrose and only 0.29 per shoot in medium containing mannitol. In vitro derived cormlets sprouted from apical and axillary buds on MS medium containing 12 mg dm⁻³ BA, 3 mg dm⁻³ indolebutyric acid and 30 g dm⁻³ sucrose. Daughter cormlet formation from in vitro derived cormlets was also observed.     

Secondary Somatic Embryogenesis in Abies numidica

The induction of secondary somatic embryogenesis in Abies numidica De Lann. was achieved. Precotyledonary, cotyledonary, and desiccated cotyledonary embryos were used as explants. Cotyledonary embryos before desiccation were the most suitable. The most beneficial was induction medium Schenk and Hildebrandt (SH) with 1 mg dm⁻³ thidiazuron and 1000 mg dm⁻³ myo-inositol. Initiation frequency was from 1 to 34 %. Maturation of somatic embryos was achieved on modified Murashige and Skoog medium supplemented with 40 g dm⁻³ maltose, 100 g dm⁻³ polyethylene glycol-4000 and 10 mg dm⁻³ abscisic acid. Mature somatic embryos after three weeks of desiccation germinated on SH medium with 10 g dm⁻³ charcoal and 10 g dm⁻³ sucrose. This revised version was published online in July 2006 with corrections to the Cover Date.     

Simultaneous Regeneration of Different Morphogenic Structures from Quince Leaves as Affected by Growth Regulator Combination and Treatment Length

Experiments were performed to evaluate the capacity of quince (Cydonia oblonga Mill.) leaves to regenerate somatic embryos and shoots and/or roots simultaneously. Leaves, treated for 2 d in liquid medium containing 2.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid were cultured for 0, 3, 6, 9, 12, 15, 18, 21 d on a gelled medium supplemented with 1 mg dm⁻³ kinetin (Kin) and 0.1 mg dm⁻³ naphthalenacetic acid (NAA) and were transferred to a medium either without growth regulator (GR-) or containing 0.6 mg dm⁻³ 6-benzylaminopurine (BA) + 0.2 mg dm⁻³ gibberellic acid (GA₃) + 0.06 mg dm⁻³ indole-3-butyric acid (IBA) (GR+). Leaves producing somatic embryos (SEs) only, or adventitious roots (Rs) only, or SEs+Rs simultaneously, were detected on GR- culture medium; on GR+ medium, leaves producing adventitious shoots (Ss) only, SEs+Ss or SEs+Rs+Ss simultaneously, also appeared. Leaves producing both Ss+Rs were never detected. Proportions among the various types of regenerating leaves varied according to both the length of Kin+NAA treatment and the presence or absence of GR in the transfer medium. The greatest variations, both on GR− and on GR+, took place within the first 9 d of culturing on Kin+NAA. After this period, no further substantial differences in the trend of each type of regenerating leaf were observed. The length of the treatment with Kin+NAA also modified the proportions between the different types of morphogenic structures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Direct organogenesis from leaf explants of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> and cultivation in bioreactor

Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog’s nutrient medium supplemented with 8.88 μM of N ⁶-benzylaminopurine and kinetin ranging from 4.65 to 6.98 μM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm⁻³ sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 μM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm⁻³ sucrose resulted in a high biomass yield of 50.68 g dm⁻³ (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 μM, 15 g dm⁻³ sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.     

<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.     

Proline Accumulation, Protein Pattern and Photosynthesis in Bacopa Monniera Regenerants Grown under NaCl Stress

Shoots of Bacopa monniera exhibited 100 % regeneration on Murashige and Skoog medium with 2 % sucrose, 0.2 mg dm⁻³ 1-naphthaleneacetic acid, 0.5 mg dm⁻³ 6-benzylaminopurine and 50 mg dm⁻³ glutamine. When the medium was supplied with various concentrations (5 - 15 g dm⁻³) of sodium chloride, proline content in regenerants was six times higher than in the control. With increasing NaCl concentration photosynthetic rate decreased and fresh mass and root length of regenerants declined. NaCl also induced formation of new proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> minimum growth for conservation of <Emphasis Type="Italic">Drosophyllum lusitanicum</Emphasis>

The present paper reports a protocol for minimum growth conservation of Drosophyllum lusitanicum (L.) Link. in vitro. Double-node cuttings were maintained for 4, 8 and 12 months at 5 or 25 °C in the dark. The effects of sucrose either alone at 5, 20, 30, 40 and 60 g dm⁻³ or at 20, 40 and 60 g dm⁻³ in combination with 20 g dm⁻³ mannitol, on survival and post-storage shoot multiplication efficiency were investigated. The cultures could effectively be conserved under minimum growth at 5 °C for 8 months on Murashige and Skoog’s medium supplemented with 60 g dm⁻³ sucrose, 20 g dm⁻³ mannitol and 0.91 μM zeatin. Following extended conservation, the cultures could be successfully regenerated into new shoots, and they were morphologically similar to those of non-stored controls.     

High efficiency organogenesis and analysis of genetic stability of the regenerants in <Emphasis Type="Italic">Solanum melongena</Emphasis>

A novel protocol for plant regeneration from cotyledon explants of eggplant (Solanum melongena) reducing concentration of sucrose was established. The most efficient bud induction medium consisted of Murashige and Skoog (MS) medium supplemented with 2.0 mg dm⁻³ zeatin, 0.1 mg dm⁻³ indoleacetic acid and 10 g dm⁻³ sucrose. After 15 d, the shoot buds were fragmented and transferred to the shoot elongation MS supplemented with 1.0–2.0 mg dm⁻³ gibberellic acid and 4.0–8.0 mg dm⁻³ AgNO₃, which promoted shoots elongation. The genetic stability of the regenerated plants was analyzed by flow cytometry, RAPD and SSR molecular markers. The results indicated that almost no somaclonal variation was detected among the regenerants.     

Somatic embryogenesis and plant regeneration of <Emphasis Type="Italic">Abelmoschus esculentus</Emphasis> through suspension culture

A simple and reliable protocol for regeneration of okra through somatic embryogenesis from suspension cultures has been developed. Embryogenic callus was obtained from hypocotyl explants cultured on media with Murashige and Skoog (MS) salts, Gamborg (B5) vitamins, 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg dm⁻³ naphthaleneacetic acid (NAA), 25 mg dm⁻³ polyvinylpyrrolidone and 30 g dm⁻³ sucrose. More number and high frequency of healthy embryoids appeared individually in suspension culture containing MS salts, B5 vitamins, 2.0 mg dm⁻³ 2,4-D and 1.0 mg dm⁻³ kinetin. Formation of cell clusters from the single cells was clearly noticed during ontogeny. Matured embryos at the cotyledonary stage were transferred to agar solidified medium for germination. The best conversion of embrya into plantlets (67.3 %) was recorded on media with half strength MS salts, B5 vitamins, 0.2 mg dm⁻³ benzylaminopurine (BAP) and 0.2 mg dm⁻³ gibberellic acid (GA₃). The plantlets were transferred to soil and hardened in the plastic pots. After proper acclimatization, the plantlets regenerated through somatic embryogenesis were compared to seed grown plants to observe any variation.     

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.     

Promotion of direct somatic embryogenesis of <Emphasis Type="Italic">Oncidium</Emphasis> by adjusting carbon sources

To further optimize a culture medium for induction of direct embryo formation of Oncidium cvs. Gower Ramsey and Sweet Sugar, five kinds of carbon sources, cellibiose, fructose, glucose, maltose and sucrose at 10, 20, 30 and 60 g dm⁻³ were tested in this study. Cellibiose supply had an inhibitory effect and resulted in high percentage of explant browning in both cultivars. By contrast, fructose, glucose and sucrose were all effective for direct embryo induction. In cv. Gower Ramsey, the suitable ranges of concentration were found at 30–60 g dm⁻³ of sucrose, 10–20 g dm⁻³ of glucose and 20–30 g dm⁻³ of fructose, respectively. The suitable ranges for cv. Sweet Sugar were at 20–60 g dm⁻³ of sucrose, 10–30 g dm⁻³ of glucose, 10–20 g dm⁻³ of fructose and 30–60 g dm⁻³ of maltose, respectively. The highest amount of embryos was obtained at 30 g dm⁻³ of sucrose for cv. Gower Ramsey and at 20 g dm⁻³ of glucose for cv. Sweet Sugar.     

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm⁻³ α-naphthalene acetic acid (NAA) + 0.5 mg dm⁻³ 6-benzyladenine (BA) for embryos production and 0.03 mg dm⁻³ NAA + 0.5 mg dm⁻³ BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.     

Extracellular Matrix in Early Stages of Direct Somatic Embryogenesis in Leaves of Drosera spathulata

The growth and water relations of Paulownia fortunei in photoautotrophic cultures (nutrient medium lacking sucrose and growth regulator) with CO₂ enrichment (PWAH) or without CO₂ enrichment (PWAL) were compared with those in photomixotrophic shoot (PWC; 30 g dm⁻³ sucrose and 0.3 mg dm⁻³ N⁶-benzyladenine) and root cultures (PWR; 0.3 mg dm⁻³ indole-3-butyric acid). The photoautotrophic and photomixotrophic cultures were incubated under photosynthetic photon flux 125 and 60 μmol m⁻² s⁻¹, respectively. 100 % sprouting and significantly higher number of shoots (1.6) were obtained with PWAH as compared to PWAL and PWC. PWAH and PWAL stimulated spontaneous rooting from the cut end of axillary shoots. In PWAH, 84 % of shoots rooted with an average of 5.9 roots per shoot and 4.0 cm of root length in 21 d. Rooting of photomixotrophic shoot cultures were stimulated by an auxin treatment. In this case, 98.3 % of shoots were rooted with an average of 4.6 roots per shoot and 1.9 cm length. A microscopic observation on leaf abaxial surface prints from photomixotrophic shoot and root cultures showed widely open (6 – 8 μm) spherical stomata (12 – 14 μm) and from photoautotrophic cultures elliptical stomata (10 – 12 μm) with narrow openings (3 – 4 μm). Leaves from photomixo-trophic cultures had higher stomatal index as compared to photoautotrophic cultures. The rate of moisture loss from detached leaves was not varying significantly in different cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of selected carbohydrates on rhizogenesis of shoots saucer magnolia in vitro

Nodal segements were taken from juvenile shoots of mature 100 year-old trees of saucer magnolia (Magnolia x soulangiana Soul.-Bod.) and cultured on Standardi and Catalano medium supplemented with 1.33 μmol·dm⁻³ BA, 0.54 μmol·dm⁻³ NAA, 58 μmol·dm⁻³ sucrose and 6.0 g·l⁻¹ agar-agar. After 8 weeks, separated shoots were transferred to rooting medium with half-strength macronutrients (basal medium) supplemented with 0.3% activated charcoal and one of carbohydrates: arabinose, cellulose, fructose, galactose, glucose, lactose, mannose, rhamnose, ribose, sorbose, sucrose or xylose at 20 g·dm⁻³ and 7.0 g·dm⁻³ agar-agar. After 13 weeks of culture, shoot number, fresh and dry weight of shoots and roots, total root length and number of roots/per shoot were recorded. Percentages of rooted shoots were calculated. Fructose, mannose and xylose were the most effective carbon source on shoot proliferation followed by sucrose. The rooting response was induced by cellulose and xylose. Arabinose, rhamnose and sorbose inhibited root formation. The number of adventitious roots produced per shoot was stimulated by cellulose and xylose. Total biomass (shoot plus roots) of the plantlets was the highest at fructose and cellulose.     

Successful micropropagation protocol of <Emphasis Type="Italic">Piper methysticum</Emphasis>

An efficient in vitro propagation of kava (Piper methysticum) was established. Utilizing 15-d-old tender shoots from dormant auxiliary buds as explants, significant induction of vigorous aseptic cluster shoots was achieved in Murashige and Skoog (MS) medium containing 0.5 mg dm⁻³ 6-benzyladenine (BA), 0.5 mg dm⁻³ indole-3-acetic acid (IAA), and antibiotics after 30 d. In vitro rooting was achieved at 100 % efficiency in MS medium containing 0.75 to 1.00 mg dm⁻³ IAA or indole-3-butyric acid and 3 % sucrose. The most robust and long roots were observed in medium with IBA. Moreover, the embryonic callus was induced from petioles in MS medium supplemented with 1.0 mg dm⁻³ BA and 0.1 mg dm⁻³ IAA, of which 70 % differentiated into shoots in the presence of 1.0 mg dm⁻³ BA and 0.5 mg dm⁻³ IAA.     

Somatic embryogenesis and plant regeneration through leaf culture in <Emphasis Type="Italic">Arachis glabrata (Leguminosae)</Emphasis>

Plants of two accessions of Arachis glabrata were regenerated via somatic embryogenesis. Embryogenic calli were initiated from leaflet explants on Murashige and Skoog medium supplemented with picloram alone or picloram in combination with 6-benzylaminopurine. Leaflets of accession A6138 induced the highest percentage of somatic embryos in media composed of 10 mg dm⁻³ and 15 mg dm⁻³ picloram. In contrast, 5 mg dm⁻³ picloram with 0.1 mg dm⁻³ 6-benzylaminopurine was one of the most effective combinations in accession AF385. MS medium supplemented with 2 g dm⁻³ activated charcoal (AC) used for 30 days was the most effective for embryo maturation. After 20 days of culture on MS medium devoid of growth regulators, 6 % of embryos converted into plantlets in accession A6138.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Spilanthes acmella</Emphasis>

Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm⁻³ 6-benzyladenine (BA) and 0.1 mg dm⁻³ α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm⁻³ BA and 1.0 mg dm⁻³ IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm⁻³ IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.     

Leaf ultrastructure, photosynthetic rate and growth of myrtle plantlets under different in vitro culture conditions

The in vitro rooting of myrtle (Myrtus communis L.) plantlets was performed in containers with gas permeable (V) and non-permeable (C) closures characterized by a different number of gas exchanges (1.4 and 0.3 h^{− 1}, respectively). The rooting was induced on Perlite, soaked with half strength Murashige and Skoog (MS) medium with 0.5 mg dm⁻³ IBA, either with and without 15 g dm⁻³ of sucrose. During the rooting phase, it was demonstrated that C cultures without sucrose (C−) negatively affect the growth of myrtle plantlets. The net photosynthetic rate and the starch content showed the lowest values in C cultures with and without sucrose (C+ and C−) while chlorophyll a content did not vary among treatments, therefore it could not be considered an indicative parameter to evaluate the autotrophic metabolism in myrtle plantlets. Electron microscopy and image analysis were employed to evaluate the leaf ultrastructure at three sample dates. Plantlet rooted in vented vessels with and without sucrose (V+ and V−) showed chloroplasts with numerous starch inclusions, while several osmiophilic plastoglobules (frequently related with leaf senescence) were found in chloroplast of leaf cells of C− myrtle plantlets.     

Direct organogenesis in hop - a prerequisite for an application ofA. tumefaciens-mediated transformation

The regeneration ability of primary explants derived from mericlones of two commercial Bohemian hops was investigated. It was found that these hops are able to regenerate shoots by direct organogenesis on media containing BAP or zeatin at concentrations 0.5–2 mg dm⁻³. The highest regeneration of shoots was achieved from either petioles or internodes at frequencies 21% and 52%, respectively, on the medium containing zeatin (2 mg dm⁻³), while relatively low amount of regenerated shoots (1.3%) was observed for leaf blade explants. On the other hand, more efficient rooting occurred on the leaf blades then on other explants. A similar pattern of regeneration we observed for HLVd-infected mericlones of clone Osvald 31 even though viroid concentration inin vitro cultures was about 8-fold higher than in field-grown plants and was 31.1 pg mg⁻¹ of fresh mass in the average. These results suggest that HLVd infection did not impair organogenesis. We found that high 2,4-D concentration pretreatment (11 mg dm⁻³) did not promote somatic embryogenesis. Although this treatment suppressed direct organogenesis, the inhibition was not complete and in low frequency the shoot regeneration was seen. Sensitivity of hop explants to antibiotics commonly used inAgrobacterium-mediated transformation was assayed. It was found that kanamycin (100–200 mg dm⁻³) suppressed efficiently callogenesis, root formation and shoot proliferation. An estimation of effect of kanamycin (200 mg dm⁻³) and ticarcillin (500 mg dm⁻³) on morphogenesis was performed using regeneration medium. The inhibitory effects observed suggest that these conditions could be used inAgrobacterium transformation/selection system. Communicated by J. TUPY