<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.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.     

<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> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

Shoot regeneration and free-radical scavenging activity in <Emphasis Type="Italic">Silybum marianum</Emphasis> L.

The morphogenic potential and free-radical scavenging activity of the medicinal plant, Silybum marianum L. (milk thistle) were investigated. Callus development and shoot organogenesis were induced from leaf explants of wild-grown plants incubated on media supplemented with different plant growth regulators (PGRs). The highest frequency of callus induction was observed on explants incubated on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ 6-benzyladenine (BA) after 20 days of culture. Subsequent transfer of callogenic explants onto MS medium supplemented with 2.0 mg l⁻¹ gibberellic acid (GA₃) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) resulted in 25.5 ± 2.0 shoots per culture flask after 30 days following culture. Moreover, when shoots were transferred to an elongation medium, the longest shoots were observed on MS medium supplemented with 0.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, and these shoots were rooted on a PGR-free MS basal medium. Assay of antioxidant activity of in vitro and in vivo grown tissues revealed that significantly higher antioxidant activity was observed in callus than all other regenerated tissues and wild-grown plants.     

Somatic embryogenesis and plant regeneration from leaf and petiole explants of <Emphasis Type="Italic">Campanula punctata</Emphasis> Lam. var. <Emphasis Type="Italic">rubriflora</Emphasis> Makino

A simple and efficient protocol was developed for somatic embryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L⁻¹ 6-benzyladenine (BA). On this medium, a mean number of 19.5 and 31.2 SE were developed per leaf and petiole explants, respectively. Embryos were induced both light and dark conditions but culturing the explants 2 weeks in the dark followed by 3 weeks under light resulted in high frequency of embryo formation. Globular embryos germinated best on MS medium supplemented with 0.3% (w/v) activated charcoal (AC) and 1.0 mg L⁻¹ GA₃. The germinated plantlets grew further on MS medium containing 0.3% AC. Plantlets were successfully acclimatized in the greenhouse with 94% survival rate. This is the first report on induction of somatic embryogenesis in this genus and also has implications for genetic transformation, and mass clonal propagation.     

High-frequency in vitro propagation of the endangered species <Emphasis Type="Italic">Tuberaria major</Emphasis>

A novel protocol suitable for the micropropagation of the endangered species Tuberaria major using seedlings as explants is reported. Using this protocol, we studied the effects of explant type (apical shoots and nodal segments) and cytokinins [6-benzyladenine (BA), kinetin, and zeatin (ZEA)] on shoot proliferation. Explant type significantly influenced the proliferation frequency and mean number of shoots, with nodal segments showing a higher proliferation capacity. The mean number of shoots was significantly higher when the explants were cultured in half-strength (1/2) MS medium supplemented with 0.2 mg l⁻¹ BA (6.83 ± 0.77 shoots) or ZEA (6.55 ± 0.71 shoots). The shoots showed a great rooting capacity that was significantly influenced by the concentration of MS macronutrients but not by the concentration of auxins. The highest rooting frequencies (97–100%) were obtained in 1/2 MS medium with or without plant growth regulators. The plants obtained were easily acclimatized to ex vitro conditions, with 97% surviving after 6 weeks. The micropropagated plants were successfully reintroduced into their natural habitat and exhibited normal development. In conclusion, our culture protocol, with efficient seed germination, subsequent multiplication of nodal explants using ZEA at 0.2 mg l⁻¹, and successful ex vitro establishment of well-rooted plantlets on 1/2 MS medium, provides a simple and reliable methodology for the large-scale propagation of T. major, thereby contributing to germplasm preservation of this endangered species.     

Callus induction and plant regeneration in <Emphasis Type="Italic">Dorem ammoniacum</Emphasis> D., an endangered medicinal plant

Dorema ammoniacum D. Don. (Apiaceae), a native medicinal plant in Iran, is classified as a vulnerable species. Root, hypocotyl, and cotyledon segments were cultured on Murashige and Skoog (MS) (1962) medium supplemented with either 2,4-dichlorophenyoxyacetic acid (2,4-D) or naphathalene acetic acid (NAA), at 0–2 mg l⁻¹, alone or in combination with either benzyladenine (BA) or kinetin (KN), at 0–2 mg l⁻¹ for callus induction. The best response (100%) was observed from root segments on MS medium containing 1 mg l⁻¹ NAA and 2 mg l⁻¹ BA. The calli derived from various explants were subcultured on MS medium supplemented with BA (1–4 mg l⁻¹) alone or in combination with NAA or indole-3-butyric acid (IBA), at 0.2 or 0.5 mg l⁻¹ for shoot induction. Calli derived from hypocotyl segments showed significantly higher frequency of plantlet regeneration and number of plantlets than the calli derived from root and cotyledon segments. Therefore, MS medium supplemented with 2 mg l⁻¹ BA and 0.2 mg l⁻¹ IBA produced the highest frequency of shoot regeneration (87.3%) in hypocotyl-derived callus. The optimal medium for rooting contained 2.5 mg l⁻¹ IBA on which 87.03% of the regenerated shoots developed roots with an average number of 5.2 roots per shoots within 30 days. These plantlets were hardened and transferred to the soil. The described method can be successfully employed for the large-scale multiplication and conservation of germplasm this plant.     

A rapid system for micropropagation of <Emphasis Type="Italic">Swertia chirata</Emphasis> Buch-Ham. ex Wall.: an endangered medicinal herb via direct somatic embryogenesis

An improved method of direct somatic embryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness to induce somatic embryos. Leaf explants with abaxial side in the medium produced maximum number of somatic embryos. This system omits the callus stage and thus reduces the process of SE in S. chirata by 35–45 days. Embryos at different stages of development were observed. Maturation of heart stage embryos were observed on Murashige and Skoog (MS) medium containing 1 mg L⁻¹ 2,4-D. Upon transfer to the germination medium, they were converted to cotyledonary stage and then plantlets of 33% and 68% of them were converted to cotyledonary stage and then plantlets on MS medium supplemented with 0.05 and 0.1 mg L^-1 GA₃ respectively. The 2,4-D alone at 1.0 or 1.5 mg L⁻¹ was found to be better for embryogenic tissue initiation than 2,4-D in combination with indole-3-acetic acid or α-naphthalene acetic acid. For further embryo development, 2,4-D was combined with cytokinins such as 6-benzylaminopurine (BAP) and kinetin or plant growth regulator free medium or medium with 50% reduced concentration of the same hormone while subculturing. Mean germination and percentage of survival were maximum in the medium containing 1.0 mg L⁻¹ 2,4-D in combination with 0.1 mg L⁻¹ BAP. Regenerated plantlets were morphologically and genetically identical. This method offers a vast scope for the clonal propagation of endangered plants.     

In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.     

Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.     

Morphological evaluation and antioxidant activity of <Emphasis Type="Italic">in vitro</Emphasis>- and <Emphasis Type="Italic">in vivo</Emphasis>-derived <Emphasis Type="Italic">Echinacea purpurea</Emphasis> plants

An effective in vitro protocol for rapid clonal propagation of Echinacea purpurea (L.) Moench through tissue culture was described. The in vitro propagation procedure consisted of four stages: 1) an initial stage - obtaining seedlings on Murashige and Skoog (MS) basal medium with 0.1 mg L⁻¹ 6-benzylaminopurine, 0.1 mg L⁻¹ α-naphthalene acetic acid and 0.2 mg L⁻¹ gibberellic acid; 2) a propagation stage — shoot formation on MS medium supplemented with 1 mg L⁻¹ 6-benzylaminopurine alone resulted in 9.8 shoots per explant and in combination with 0.1 mg L⁻¹ α-naphthalene acetic acid resulted in 16.2 shoots per explant; 3) rooting stage — shoot rooting on half strength MS medium with 0.1 mg L⁻¹ indole-3-butyric acid resulted in 90% rooted microplants; 4) ex vitro acclimatization of plants. The mix of peat and perlite was the most suitable planting substrate for hardening and ensured high survival frequency of propagated plants. Significant higher levels were observed regarding water-soluble and lipid-soluble antioxidant capacities (expressed as equivalents of ascorbate and α-tocopherol) and total pnenols content in extracts of Echinaceae flowers derived from in vitro propagated plants and adapted to field conditions in comparison with traditionally cultivated plants.     

<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.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

Direct shoot organogenesis from <Emphasis Type="Italic">in vitro</Emphasis>-derived mature leaf explants of pistachio

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl⁻¹ IAA and 2 mgl⁻¹ BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl⁻¹ BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl⁻¹ of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species.     

An <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation system using callus of <Emphasis Type="Italic">Zoysia tenuifolia</Emphasis> Willd. ex Trin

Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l⁻¹ 6-benzyladenine (BA), with a frequency of 53%. Compact calli were selected and subcultured monthly on the fresh medium. Sixty-nine percent of the calli could be induced to regenerate plantlets when the calli incubated on a MS medium supplemented with 0.2 mg l⁻¹ BA under darkness. For genetic transformation, calli were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, about 12% of the callus explants produced hygromycin resistant calli on MS medium supplemented with 2 mg l⁻¹ 2,4-D, 1 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, 500 mg l⁻¹ cefotaxime after 8 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 0.2 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 46% of the resistant calli differentiated into shoots. Finally, all the resistant shoots were rooted on 1/2 MS media supplemented with 50 mg l⁻¹ hygromycin, 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. About 5% of the total inoculated callus explants produced transgenic plants after approximately 5 months. The procedure described will be useful for both, the introduction of desired genes into Z. tenuifolia and the molecular analysis of gene function.     

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.