Somatic embryogenesis and plant regeneration in açaí palm (<Emphasis Type="Italic">Euterpe oleracea</Emphasis>)

An efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from immature zygotic embryos of açaí palm (Euterpe oleracea) has been developed. Embryogenic calli (ECs) were induced from immature zygotic embryos of açaí palm on Murashige and Skoog (MS) modified medium with 2,4-dichlorophenoxyacetic acid and picloram. Embryogenic frequency was dependent on auxin type and concentration. The optimal concentration of picloram for the high-frequency induction of embryogenic calli (72%) was 225 μM. ECs were then subcultured on a differentiation and maturation medium composed of MS modified medium with 2-isopentenyladenine and naphthaleneacetic acid with subcultures at 4-week intervals. SEs were converted to plants on MS modified medium with half-strength macro- and micronutrients, 20 g l^?1 sucrose, and 2.5 g l^?1 activated charcoal and gelled with 2.5 g l^?1 Phytagel. Detailed morpho anatomical changes during the different stages of somatic embryogenesis were characterized. The development of SEs was asynchronous, and ontogenic studies confirmed that the initial cell divisions occur in the epidermal and subepidermal regions of the zygotic embryos. Broad base attachment of SEs to the epidermis indicates the presence of a suspensor.     

Somatic embryogenesis and plant regeneration in Pterocarpus marsupium Roxb.

Somatic embryogenesis (SE) has been achieved from hypocotyl-derived callus culture in Pterocarpus marsupium. Ninety percent of hypocotyl explants (excised from 12-day-old in vitro germinated axenic seedlings) produced callus on Murashige and Skoog medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid and 1 μM a 6-benzyladenine (BA). Induction of SE occurred after transfer of callus clumps (200 ± 20 mg fresh mass) to MS medium supplemented with BA at 2.0 μM, where a maximum of 23.0 ± 0.88 globular stage embryos per callus clump were observed after 4 weeks of culture. Subculturing of these embryos on MS medium supplemented with 0.5 μM BA, 0.1 μM α-naphthalene acetic acid and 10 μM abscisic acid significantly enhanced the maturation of somatic embryos to early cotyledonary stage, where 21.4 ± 0.32 embryos per callus clump were recorded after 4 weeks of culture. Of 30-well developed somatic embryos, 16.6 ± 0.33 germinated and subsequently converted into plantlets on half-strength MS medium supplemented with 1.0 μM BA. The morphologically normal plantlets with well-developed roots were first transferred to 1/4-liquid MS medium for 48 h and then to pots containing autoclaved soilrite and acclimatized in a culture room. Thereafter, they were transferred to a greenhouse, where 60% of them survived.     

Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshurica Rupr.)

Using mature cotyledonary explants of Fraxinus mandshurica, an efficient plant regeneration system was developed via somatic embryogenesis. More than 67 % of mature cotyledons of zygotic embryos yielded 23–159 somatic embryos (SEs) per explant when incubated on medium consisting of half-strength Murashige and Skoog (MS) salts and vitamins (MS1/2) supplemented with 8.88 μM 6-benzyladenine (BA), 26.84 μM naphthaleneacetic acid (NAA), 75 g L^?1 sucrose, and 400 mg L^?1 casein hydrolysate (CH). Approximately, 82 % of induced SEs were observed on browning cotyledonary explants. Histological studies of cotyledon explants at various stages of somatic embryogenesis revealed that the SEs originated from single epidermal cells and developed to the globular, heart, torpedo, and cotyledonary stage embryos. Secondary somatic embryos (SSEs) formed on the surface of radicle tips of the SEs. Addition of low concentrations of NAA and 200–400 mg L^?1 CH to MS1/2 medium increased SSE induction. Cotyledonary SSEs were cultured on MS1/2 medium with 10 mM abscisic acid in the presence of light to promote maturation, and >92 % of mature SSEs were able to germinate with normal shoots. After 8 weeks in culture in the presence of light on medium with one-third of the MS macroelements as well as 0.06 μM NAA, >94 % of the germinated SSEs converted into plantlets. Plantlets acclimatized successfully to ex vitro conditions and developed normal phenotypes under field conditions.     

Somatic embryogenesis of <Emphasis Type="Italic">Merwilla plumbea</Emphasis> (Lindl.) Speta

A simple and efficient system was developed for rapid somatic embryogenesis from leaf explants of Merwilla plumbea, a traditional but threatened medicinal plant in South Africa. Friable embryogenic callus (FEC) was obtained from leaf explants on embryogenic callus induction medium containing agar-solidified Murashige and Skoog (MS) salts and vitamins, 8.3 μM picloram, 2.3 μM thidiazuron (TDZ) and 20 μM glutamine. FEC was subsequently incubated in embryogenic callus proliferation medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.1 μM picloram for 7 days before it was transferred to liquid somatic embryo medium (SEM_L) containing MS medium supplemented with 0.4 μM picloram and 0.9 μM TDZ. In SEM_L supplemented with 150 mg L⁻¹ haemoglobin, 5.4–35.6 somatic embryos per settled cell volume of 500 mg FEC were obtained. These embryos were at globular to cotyledonary developmental stages. Embryo maturation, germination and plant formation rate was 94.4% following transfer of SEs to half-strength MS medium supplemented with 1.4 μM gibberellic acid. Plantlets transferred into soil acclimatized in the misthouse and established successfully in the greenhouse (100%). This is the first report on induction of Merwilla plumbea somatic embryogenesis. The protocol developed offers controlled vegetative propagation by alleviating extinction threats, ensures germplasm conservation and provides a system for physiological, biochemical, molecular and cellular studies of embryo development.     

In vitro propagation via organogenesis and embryogenesis of <Emphasis Type="Italic">Cyrtanthus mackenii</Emphasis>: a valuable threatened medicinal plant

Efficient and simple, organogenesis (direct and indirect) and somatic embryogenesis (cell suspension) systems were developed for in vitro propagation of Cyrtanthus mackenii, a valuable economic plant from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of sucrose, plant growth regulators (PGRs), glutamine, phloroglucinol (PG) and 6-(2-hydroxy-3-methylbenzylamino) purine (PI55). MS medium solidified with 8 g L^?1 agar (MS_S) containing 40 g L^?1 sucrose, 10 µM picloram, 2.5 µM benzyladenine (BA) and 20 µM glutamine produced a higher number of shoots from white nodular callus. This was however, not significantly different to direct shoot regeneration on media containing 10 µM picloram, 2.5 µM BA and a reduced concentration of sucrose and glutamine. The regenerated shoots were rooted best with MS_S medium incorporating 10 µM PG. The number of somatic embryos (SEs) were significantly higher using liquid MS medium containing 30 g L^?1 sucrose, 0.5 µM picloram, 1 µM thidiazuron or BA and 3 µM glutamine or gibberellic acid. The embryos were germinated in PGR-free MS_S medium. All plantlets were successfully acclimatized in the greenhouse. Histological studies confirmed the different developmental stages and bipolar structure of SE. The organogenesis and somatic embryogenesis protocols provides a system for large scale propagation and germplasm conservation. Developed protocols can be used for clonal production and pharmacological and genetic transformation studies.     

Somatic embryogenesis in banana (Musa acuminata AAA cv. Grand Naine): effect of explant and culture conditions

An improved, rapid, reproducible, and simple protocol has been developed for somatic embryogenesis in banana cv. ‘Grand Naine’ using explants derived from actively growing multiple shoot cultures. Many restrictive factors remain in banana embryogenesis such as long duration, unpredictability, and a high degree of genotype dependence. In the present study, we used split shoot tips from 4-wk-old cultures as explants. Somatic embryos were induced in 15 d directly in Murashige and Skoog (MS) medium supplemented with different combinations of 0–8.28 μM picloram and 0.22–4.44 μM 6-benzylaminopurine (BA) without callus formation. Maximum embryo induction (100%) occurred when 4.14 μM picloram and 0.22 μM BA were used. Conversion of somatic embryos into plantlets occurred sporadically (2–3%) in MS medium containing α-naphthalene acetic acid (NAA; 0.53–2.68 μM) together with BA (2.22–44.39 μM), or thidiazuron (4.54 μM) plus glutamine (200 mg/L). This protocol is far superior to those already reported for fast and high frequency induction of somatic embryo. In liquid agitated culture, individual embryos separated easily and produced a large number of secondary embryos within 10 d which, upon transfer to filter paper overlaid on MS liquid medium supplemented with 4.44 μM BA, resulted in conversion (3%) into plantlets.     

New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 g?L^?1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at half-strength concentrations supplemented with 20 g?L^?1 sucrose, 2.5 g?L^?1 activated charcoal, and 2.5 g?L^?1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge.     

In vitro morphogenic response in cotyledon explants of Semecarpus anacardium L.

Three different morphogenic responses??caulogenesis, direct somatic embryogenesis, and callusing??were noted in cotyledon explants of Semecarpus anacardium L. cultured in woody plant medium (WPM) containing thidiazuron (TDZ). Thidiazuron, at all concentrations tested, induced organogenic as well as embryogenic responses. The organogenic buds differentiated to shoots and the embryogenic mass (EM) gave rise to globular embryos which differentiated up to cotyledon-stage embryos on repeated culture in growth regulator (GR)-free WPM medium containing 0.2% activated charcoal after the removal of TDZ. The organogenic and embryogenic responses were optimal in 9.08???M TDZ after the removal of TDZ. Elongated shoots rooted in half-strength liquid WPM medium with 2.46???M indole butyric acid. Plants were successfully acclimatized and transferred to soil. Histological studies confirmed the direct origin of the organogenic buds from the cotyledon explants. The EMs produced somatic embryos on repeated culture in charcoal incorporated GR-free medium. Morphogenic callus formation from the cotyledon explants was also noted. This callus on repeated culture in WPM medium with charcoal differentiated into somatic embryos. Repetitive somatic embryogenesis was evident from direct and indirectly formed primary embryos. The somatic embryos did not convert into plantlets, though sporadic germination of embryos was observed through the emergence of roots.     

Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”

Using immature embryos and cotyledons as explants, a successful immature embryo culture and efficient plant direct regeneration via organogenesis from cotyledons, which showed different patterns, was established for the “Xuemei” cultivar of Prunus mume. For immature embryo culture, high frequency plantlet forming (89.5%) from embryo axis was obtained on half-strength Murashige and Skoog (½MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). At the same time, shoots direct differentiation from cotyledons with the embryo axis development was also observed on ½MS medium containing 2.2 μM BA together with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when embryo axes were removed from cotyledons and cultured on ½MS medium supplement with 13.2 μM BA, 2.7 μM NAA (72.9%) or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA (84.2%), respectively. Regenerated shoots were successfully rooted on ½MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of embryo axes, BA and TDZ, on cotyledons’ regeneration were investigated in detail. The rooted plantlets were transferred to soil successfully with normal morphology.     

Micropropagation of the Mongolian medicinal plant <Emphasis Type="Italic">Zygophyllum potaninii</Emphasis> via somatic embryogenesis

The Mongolian medicinal plant Zygophyllum potaninii has been assessed as an endangered species with regional status. We applied the somatic embryogenesis technique using aseptic in vitro germinants of the plant as an effective propagation technology. The seed germination rate in vitro was 16.5% after 2 weeks of culture. Embryonic calli (EC) and somatic embryos (SEs) were induced using the cotyledon or hypocotyl segments of the germinants. Calli were effectively induced on MS medium supplemented with 0.1 mg/L 2, 4-dichlorophenoxy acetic acid (2, 4-d) and 0.5 mg/L 6-benzylamino purine (BA). The callus was composed of pale yellow or pale green friable cells. SE formed from EC only on Murashige and Skoog medium (MS) with 0.5 mg/L abscisic acid (ABA). Other concentrations of ABA failed to induce SE formation. All SEs germinated in MS medium with different salt levels. However, normal plant conversion was achieved only on half-strength MS medium. The converted plantlets were effectively acclimatized in vitro in sand and transferred to a mixture of sand and perlite (1:1 v/v) in the greenhouse. After 8 weeks of culture, 55.4% of the plants survived. This is a first report of propagating the medicinal desert plant Z. potaninii via somatic embryogenesis and plant regeneration.     

Somatic embryogenesis and plant regeneration in zygotic embryo explant cultures of rugosa rose

Rugosa rose (Rosa rugosa) is cultivated as a garden flower and an important genetic resource for the breeding of roses (R. hybrida). This study describes culture conditions for high frequency plant regeneration from zygotic embryo explants via somatic embryogenesis in rugosa rose. Mature zygotic embryo, cotyledon, and radicle explants formed embryogenic calluses at frequencies of 38, 6.7, and 8.8% when cultured on half-strength Murashige and Skoog medium (½MS) supplemented with 2.26, 9.05, and 9.05 μM 2,4-dichlorophenoxyacetic acid, respectively. Embryogenic calluses produced numerous somatic embryos, which then developed into plantlets on ½MS without growth regulators. Regenerated plantlets were grown to whole plants in a growth chamber.     

Repetitive somatic embryogenesis from leaves of the medicinal plant Petiveria alliacea L.

Leaf segments from in vitro-grown shoot cultures of Petiveria alliacea were incubated on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin, thidiazuron, 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). Direct somatic embryogenesis was induced in response to all tested concentrations of 2,4-D and PIC. Primary somatic embryos displayed highly repetitive embryogenesis, both on the induction medium and in liquid hormone-free MS medium. Plantlets were obtained from these secondary embryos at an estimated frequency of 5 %, after 180 days of culture on half-strength MS medium gelled with 0.2 % Phytagel. Simultaneous development of friable non-embryogenic callus was also observed on media containing PIC or 2,4-D at different concentrations. Cell suspension cultures initiated from these callus tissues did not show an increase in biomass. The embryogenic portions formed at the surface of the explants in response to 20.0 μM PIC were inoculated in hormone-free full-or half-strength liquid MS medium (MS0) and showed high rates of secondary embryogenesis, resulting in the production of a mean of 35 embryos for each embryo inoculated at the culture initiation. Embryos that started the conversion process in the liquid MS0 medium originated whole plants at a frequency of 100 % when transferred to MS0 medium solidified with 0.7 % agar. Acclimatization was achieved in 90 % of the converted plantlets, with the production of phenotypically normal plants. This system is potentially useful for the micropropagation of this species, as well as for the production of substances with pharmacological interest, such as dibenzyl trisulfide.     

Repetitive somatic embryogenesis in carnation on picloram supplemented media

An efficient method of repetitive somatic embryogenesis and plant regeneration of two carnation cultivars (Sagres and Impulse) was established using a two-step protocol. In the first step, embryogenic tissue were induced from petal explants on MS culture medium containing 9% sucrose (w/v), 9 μM 2,4-D and 0.8 μM BA. In the second step, embryogenic tissue transferred onto the MS medium containing 3% sucrose supplemented with different concentrations of picloram (0.8, 2, 4, 8 and 16 μM) to produce primary somatic embryos. Precotyledonary clumps and cotyledonary somatic embryos were then isolated and subcultured onto the same media as the second step where they formed secondary somatic embryos in repetitive cycles. Cotyledony somatic embryos were converted into plantlets when they transferred onto the growth regulator-free half-strength MS medium followed by being acclimated to the greenhouse conditions.     

Establishment of somaclonal variants of Robusta coffee with reduced levels of cafestol and kahweol

Cafestol (caf) and kahweol (kah) are two diterpenes uniquely associated with the unsaponified lipid fraction of coffee brew and are reported to be responsible for an increase in serum cholesterol and triglyceride levels. The plant growth regulators (PGRs) indole-3-acetic acid (IAA), N ⁶-benzyladenine (BA), and thidiazuron (TDZ); the plant growth-promoting agents silver nitrate, triacontanol (TRIA), and coconut water; and some PGR antagonists such as lovastatin, paclobutrazol, and 2,3,5-triiodobenzoic acid (TIBA) were used to determine the variation of caf and kah in somatic embryos of Robusta coffee (Coffea canephora, CxR variety). Embryogenic (EG) medium was comprised of half-strength Murashige and Skoog basal components (½MS) supplemented with 2.85 μM IAA and 1.11 μM BA. After an 8-wk culture, somatic embryos were subjected to diterpene extraction and HPLC analysis of caf and kah profiles. TRIA-supplemented (5 μg L^?1) EG medium devoid of IAA reduced the levels of caf and kah by 18–24 and 48–55%, respectively, in coffee somatic embryos. Similarly, the combination of 2.85 μM IAA, 2.27 μM TDZ, and 5–10% coconut water in ½MS basal medium drastically reduced the caf and kah levels in somatic embryos. There was 60–75% reduction in both caf and kah in the presence of 5 μM TIBA, followed by 56–62% reduction in the presence of 10 μM silver nitrate. In contrast, there was 25–32% elevation of caf and kah in EG medium supplemented with 5 μM paclobutrazol. In this study, for the first time, somaclonal variants of C. canephora with reduced levels of diterpenes caf and kah were established. Furthermore, these lines exhibited consistency in their metabolite profiles when cultivated under greenhouse conditions. In-depth investigations at physiological level are warranted in order to elucidate the actual mechanism of these PGR inhibitors on alterations in endogenous pools of diterpenes in coffee somatic embryos.     

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Induction of somatic embryos and plant regeneration was demonstrated for the first time in Alnus glutinosa. Somatic embryos were initiated from zygotic embryos collected 1–3 weeks post-anthesis (WPA), i.e., when they were at globular or early cotyledonary stage and were 0.5–1 mm in length. Induction frequency (16.6 %) and the mean number of somatic embryos (4.5 embryos/explant) were highest after culture of zygotic embryos, collected at 3 WPA, on Murashige and Skoog medium (MS) supplemented with 0.9-μM 2,4-dichlorophenoxyacetic acid and 2.22-μM benzyladenine (BA). No embryogenic induction was observed on medium with BA alone. Initial somatic embryos differentiated indirectly from callus tissue formed at the surface of the zygotic embryos. Embryogenic competence was maintained by secondary embryogenesis, which was affected by explant type, plant growth regulators and genotype. Secondary embryogenesis was induced by culture of small groups of whole somatic embryos or isolated cotyledon explants on medium consisting of MS medium (half-strength macronutrients) supplemented with 0.44-μM BA. Histological study of isolated cotyledon explants revealed that secondary embryos developed directly from differentiated embryogenic tissue on the surface of cotyledons. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were evident. For plantlet conversion, somatic embryos were transferred to maturation medium supplemented with 3 % maltose, followed by 6 weeks of culture in Woody Plant Medium supplemented with 0.44-μM BA and 0.46-μM Zeatin (Z). This novel protocol appears promising for mass propagation, conservation and genetic transformation of black alder.     

Factors affecting morphogenesis from immature cotyledons of Phaseolus coccineus L.

Direct somatic embryogenesis as well as somatic embryogenesis and organogenesis mediated by small glossy calluses were obtained from immature cotyledon explants of bean (P. coccineus) cv Streamline 770 on a modified half-strength MS medium (Murashige & Skoog 1962) containing various concentrations of (2-isopentenyl)adenine and 2-naphthoxyacetic acid. Substitution of sucrose with glucose gave, in the range of concentrations tested, the strongest enhancement of the morphogenic process. Further improvement regarding the number of morphogenic cotyledons, the number of regenerations per cotyledon and the quality of the embryos was observed when 2,3,5-triiodobenzoic acid or abscisic acid were added to the medium. After cycles of micropropagation on MS medium plus 4.4 M 6-benzyladenine and rooting in the absence of growth factors, plantlets were adapted to ex vitro conditions and grown to maturity.Abbreviations ABA abscisic acid - AC activated charcoal - BA 6-benzyladenine - 2,4-D (2,4-dichlorophenoxy)acetic acid - DHZ dl-dihydrozeatin - IAA indole-3-acetic acid - 2iP (2-isopentenyl)-adenine - NAA 1-naphthaleneacetic acid - NOA 2-naphthoxyacetic acid - PBA N-benzyl-9-(2-tetrahydropyranyl)adenine - PIC picloram - PVP polyvinylpyrrolidone - TDZ thidiazuron - TIBA 2,3,5-triiodobenzoic acid - ZEA zeatin     

In vitro propagation, proscillaridin A production and antibacterial activity in Drimia robusta

Drimia robusta is a threatened traditional medicinal plant extensively used in South Africa. Rapid in vitro mass propagation of the species was developed for commercial cultivation from leaf explants using various concentrations and combinations of plant growth regulators and organic elicitors. The highest number of regenerated shoots per explant (14.6 ± 0.54) was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 2.27 μM thidiazuron (TDZ), 2.22 μM benzyladenine (BA) and 20 μM glutamine. Adventitious shoots were rooted and the plantlets were successfully acclimatized (100 %) in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Proscillaridin A (PsA) content and the antibacterial activity of in vitro and ex vitro regenerated plants were evaluated in different tissues in comparison to naturally-grown plants. The highest content of PsA (19.68 μg mg^?1 DW) was recorded in roots of ex vitro plants which were grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 100 mg l^?1 casein hydrolysate. In vitro regenerated plants grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 50.8 μM MBZ gave high antibacterial activity (MIC of 0.156 mg ml^?1) against both Gram-positive and Gram-negative bacteria. Using this protocol the regenerated plants can be used in traditional medicine as an alternative to naturally collected plants.     

Somatic embryogenesis and shoot regeneration from leaf derived callus of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh

Abstract

The development of in vitro regeneration systems for Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh, a medicinal plant used for treating neurological disorders, is described. For the first time in this variety, somatic embryogenesis and shoot regeneration were induced from leaf-derived callus. Well-formed plantlets were obtained through both shoot regeneration and somatic embryogenesis, with separate morphogenetic programmes. Proembryogenic masses were obtained in liquid MS and B5 media supplemented with 5.8 μM 2,4-D, 1.34 μM NAA, and 1.16 μM Kin; after being transferred onto hormone-free medium, they formed whitish and spherical structures that subsequently developed into the heart and torpedo stages.

On MS agarized medium containing thidiazuron (TDZ) at different concentrations (3, 6, 9, 12 μM) combined with 2 μM IBA, only shoot regeneration, and not somatic embryogenesis, was obtained. The mean number of shoots increased significantly when the concentration of TDZ was 3 μM.     

Micropropagation of Semecarpus anacardium L. from mature tree-derived nodal explants

A protocol was established for micropropagation of Semecarpus anacardium L. from mature tree-derived twigs. Sixty percent of aseptic cultures were obtained by surface sterilization with Bavistin, liquid detergent, and cefotaxime. Elongated twigs collected before flowering were optimum for in vitro culture initiation. Meristematic activity was triggered at all concentrations of thidiazuron (TDZ) incorporated into Woody Plant Medium. TDZ suppressed elongation of axillary buds, resulting into swollen meristems and upon its elimination multiple shoot primordia formation and differentiation were noted. Differentiation and shoot elongation were slower in explants pre-cultured with higher concentrations of TDZ. Swollen axillary meristems pre-cultured on TDZ (9.08 and 13.62 μM) failed to differentiate, whereas TDZ at 2.27 μM was optimal for shoot differentiation and elongation. Multiple bud induction was favored by 4.45 μM of TDZ. Differentiation of multiple shoot primordia by repeated subculturing on growth regulator-free medium and rooting was 100% in filter-paper supported half-strength liquid medium containing 7.38 μM IBA. Rooting was 90% in shoots placed directly in half-strength liquid medium with 2.46 μM IBA. Rooted plantlets hardened in soil:sand mixture (1:1) were transferred to green house. Genetic uniformity of in vitro raised clones with mother plant was confirmed by Inter-Simple Sequence Repeat markers.     

Plant regeneration from suspension cells induced from hypocotyls derived from interspecific cross Alstroemeria pelegrina × A. magenta and transformation with Agrobacterium tumefaciens

Embryogenic cell suspension cultures were established using the ovule culture of an interspecific cross, Alstroemeria pelegrina var. rosea × A. magenta. Ovules harvested 14 days after pollination were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGRs); calli were produced on the hypocotyl surface in germinating zygotic embryos. Suspension cells were induced from the calli by using liquid MS media containing 2,4-dichlorophenoxyacetic acid or 4-amino-3,5,6-trichloropyridine-2-carboxylic acid (picloram). Adventitious embryos developed from the suspension cells on half-strength MS medium supplemented with 0.5 mg l⁻¹ of both α-naphthaleneacetic acid and N⁶-benzylaminopurine; they grew into plantlets on the same medium. The plantlets formed rhizomes following transfer to half-strength MS medium without PGRs, and acclimatized plants were easily established. Subsequently, Agrobacterium-mediated transformation system was applied. The suspension cells were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which contain neomycin phosphotransferase II, hygromycin phosphotransferase and intron-containing ?-glucuronidase (intron-GUS) genes. Seven days after co-cultivation, the cells were subjected to GUS assay; staining was most pronounced in the cells subcultured in a picloram-containing liquid medium and co-cultivated with EHA101/pIG121Hm. The co-cultivated cells were transferred to the MS medium containing picloram and 20 mg l⁻¹ hygromycin; 1 month later, several hygromycin-resistant callus lines showing GUS activity were obtained. Transgenic plants were obtained through our plant regeneration system, and foreign gene insertion into the regenerated plants was confirmed by polymerase chain reaction.