Efficient organogenesis and plantlet regeneration in the timber species <Emphasis Type="Italic">Cunninghamia lanceolata</Emphasis> (Lamb.) Hook

Two efficient regeneration systems were developed in Cunninghamia lanceolata, the most important conifer for industrial wood production in China. Cotyledons and hypocotyls derived from greenhouse-grown seedlings were used as initial explants in our research. A high frequency (95.1?±?1.84%) of adventitious buds were initiated directly from cotyledons cultured on Douglas-fir cotyledon revised (DCR) medium supplemented with 1 mg l^?1 benzyladenine (BA), 0.1 mg l^?1 α-naphthaleneacetic acid (NAA), and 0.004 mg l^?1 thidiazuron (TDZ) with a maximum mean number of adventitious buds per cotyledon explant of 3.76?±?0.08. In contrast, a high percentage (93.73?±?0.55%) of adventitious buds regenerated via callus produced from hypocotyls cultured on DCR medium supplemented with plant growth regulators with a maximum number of adventitious buds per explant (16.71?±?0.34). Adventitious buds elongated on DCR medium supplemented with 0.2 mg l^?1 BA and 0.02 mg l^?1 NAA. After liquid pretreatment with 50 mg l^?1 indole-3-butyric acid (IBA), over 95% of the shoots successfully rooted on ½ DCR medium supplemented with 0.3 mg l^?1 IBA. The innovated systems reported in this study will be useful tools for future genetic manipulation of C. lanceolata and may be adapted for large-scale propagation in other conifers.     

Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana)

Opening of apical and axillary buds of mature Douglas-fir and sugar pine trees was obtained on a newly formulated basal medium (DCR) without growth regulators. Elongation of buds was observed on 1/2 strength DCR with 0.3% activated charcoal (DCR-1). In sugar pine, multiple shoots were obtained when explants on DCR with 0.5 mg/1 BAP for 5–6 weeks were transferred to DCR-1 medium. On subculture, axillary buds again developed when shoots were cultured on DCR with 0.2 mg/1 BAP for Douglas-fir and 0.5 mg/1 BAP for sugar pine. These buds were again elongated on DCR-1 medium. By subculturing 7–10 shoots of Douglas-fir and 2–3 shoots of sugar pine, over 100 shoots can be obtained in a year.Abbreviations BAP N⁶-benzylaminopurine - KN kinetin - NAA -naphthalene acetic acid - IAA Indole-3-acetic acid - MS Murashige-Skoog medium - WPM woody-plant medium     

Plant regeneration from callus cultures of Psoralea corylifolia Linn

Plantlet regeneration via organogenesis was achieved in callus cultures derived form mature leaves, stems and leaves, petioles and roots of young seedling of Psoralea corylifolia on Murashige and Skoog medium supplemented with 2.5–3.0 mg L^-1 BA, 1.0 mg L^-1 NAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more readily from juvenile explants (seedling source) as compared to the mature explants. Addition of adenine sulphate (5 mg L^-1) to the culture medium increased the growth of shoot buds. Optimum responses were obtained in hypocotyl and leaf explants using NAA in combination with BA, the highest rate of shoot bud regeneration being in hypocotyl explants. Rooting was readily achieved on the differentiated shoots on MS basal media without growth regulators. Regenerated plantlets were successfully established in the greenhouse.     

Direct organogenesis and plantlet regeneration from mature zygotic embryos of masson pine (Pinus massoniana L.)

Mature zygotic embryos of masson pine were cultured as initial explants to investigate the process of direct organogenesis. Adventitious buds were initiated on DCR medium (Douglas-fir cotyledon revised medium) supplemented with 0.5 mg l⁻¹ N⁶-benzyladenine (BA) and 0.05 mg l⁻¹ indolebutyric acid (IBA) or α-naphthaleneacetic acid (NAA). The highest induction frequency of adventitious buds was 99.3%. Subsequent transfer of buds to medium with lower concentrations of plant growth regulators in time was necessary for differentation of high quality adventitious buds. After culturing on elongating medium, in which the proportion of cytokinins to auxins was reduced, shoots higher than 2 cm were transferred for root induction to GD medium with half of the concentration of macro-salts (½ GD) and with 2 mg l⁻¹ IBA and 0.05 mg l⁻¹ BA. The average root frequency was over 70%. After adventitious roots had appeared, the shoots were transferred to ½ GD medium with a lower concentration of IBA (0.2 mg l⁻¹) for further root development.     

An efficient callus-based in vitro regeneration protocol for Warburgia Ugandensis Sprague,an important medicinal plant in Africa

Warburgia ugandensis Sprague is a woody species in the family Canellaceae and an important source of medicines in Africa. Natural propagation of W. ugandensis is problematic due to its recalcitrant seeds and lack of an efficient in vitro regeneration system for this species. This study describes an efficient regeneration protocol. Petiole bases and shoot tips were used as explants. Callus tissue developed when the explants were cultured on Murashige and Skoog medium containing 30 g L⁻¹ sucrose and 7 g L⁻¹ agar (MS30 medium), supplemented with 1.0 mg L⁻¹ indole-3-butyric acid (IBA), 1.6 mg L⁻¹ 6-benzylaminopurine (BA), and 0.1 mg L⁻¹ thidiazuron (TDZ). Adventitious buds were efficiently induced from the callus when the MS30 medium was supplemented with 0.8 mg L⁻¹ BA and 0.2 mg L⁻¹ IBA. Root induction occurred within 7–10 d on half-strength MS30 medium supplemented with 0.8–1.0 mg L⁻¹ 1-napthalene acetic acid (NAA), 0.2 mg L⁻¹ IBA, and 0.03% (w/v) activated charcoal (AC). Roots were followed by root elongation on the same medium but lacking NAA and IBA. Approximately 50% of the plantlets cultured produced roots, while more than 80% of the plantlets survived and successfully grew to maturity.

     

Identification of somaclonal variants in proliferating shoot cultures of Senecio cruentus cv. Tokyo Daruma

A protocol for in vitro propagation of cineraria (Senecio cruentus) was developed. The highest frequency of shoot proliferation was obtained from nodal explants cultured on Murashige and Skoog (MS) medium supplemented with 2.0?mg L^?1 6-benzyladenine (BA) and 0.5?mg L^?1 ??-naphthalene acetic acid (NAA), with a mean number of 14 shoots per explant. A high concentration of BA (4.0?mg L^?1) and repeated subcultures resulted in hyperhydric shoots. Decreasing the BA concentration to 1.0?mg L^?1 in the culture medium eliminated hyperhydricity. The concentration of ammonium nitrate (NH₄NO₃) and temperature had marked effects on somaclonal variation. Variation was observed when the cultures were maintained at 15?°C but not at 25?°C. Variants with blue-colored leaves and stems were identified; whereas, normal plants maintained their green-colored leaves and stems. The highest frequency of variation (67.5?%), with a mean number of 3.0 variant shoots per explants, was obtained on shoot proliferation medium (MS?+?2.0?mg L^?1 BA and 0.5?mg L^?1 NAA) devoid of NH₄NO₃. The best rooting (100?%), with the highest number of roots per shoot (10.8) and the greatest root length (6.8?cm) was obtained on medium supplemented with 0.1?mg L^?1 NAA. In vitro-grown plantlets were successfully acclimatized in a greenhouse, and transferred to the field.     

Plantlet regeneration from mature zygotic embryos and embryonic explants of masson pine （Pinus massoniana Lamb.）

Excised zygotic embryos,cotyledons and hypocotyls of juvenile seedlings of masson pine were grown on DCR medium supplemented with several concentrations of various plant phytohormones.BA(1.0mg/L) in combination with NAA(0.05mg/L) in DCR medium was found to increase the formation of adventitious buds from mature zygotic embryos,but most of them were formed at the tips of embryonic cotyledons.Adventitious buds were obtained from cotyledons and hypocotyls from juvenile seedlings when they were cultured on DCR medium containing BA 3-5 mg/L and NAA 0.1-0.2 mg/L.Elongation of buds were observed on hormone-free DCR medium with or without activated charcoal(0.5%).Root initiation was achieved with full or half strength DCR medium supplemented with IBA 1.0 mg/L and NAA 0.25-0.5 mg/L.Approximately 11-20 axillary buds formed on each explant when juvenile seedling explants were treated(3-20h) with BA 50-100 mg/L,followed by transfer to hormone-free DCR medium.The maximum number of shoots obtained per explant within six months was 33.     

杉木再生系统的比较研究

通过对杉木(Cunninghamia lanceolata Hook)再生系统的比较研究,建立了子叶、下胚轴、茎段和针叶等四个再生体系。子叶在附含1mg/L BA和0.1mg/L NAA的DCR培养基中再生频率为93.7%±0.45%,平均芽数为3.76±0.25;下胚轴在附含2mg/L BA和0.2mg/L NAA的DCR培养基中再生频率为96.2%±0.35%,平均芽数为17.4±0.18;茎段在附含1mg/L BA和0.1mg/L NAA的DCR培养基中再生频率达100%,平均芽数为3.28±0.11;针叶在附含1.5mg/L BA和0.1mg/L NAA的DCR培养基中再生频率达84.5%±0.45%,平均芽数仅为1.42±0.08。不定芽在附含0.2mg/L BA和0.02mg/L NAA的DCR培养基中有效伸长。芽苗经预处理后在附含0.3mg/L IBA的1/2 DCR培养基中生根效果最佳。     

Efficient plant regeneration from in vitro leaves and petioles via shoot organogenesis in Sapium sebiferum Roxb.

Sapium sebiferum Roxb. is a widespread and economically important multipurpose tree due to its high value in ornamental, and biodiesel production as well as medicine. A highly efficient in vitro plant regeneration system through direct shoot organogenesis was established for the first time from leaves and petioles of S. sebiferum. The results showed that plant growth regulators (PGRs), mechanical damage, explant orientation, explant source, and developmental stage had a strong influence on the in vitro morphogenesis of S. sebiferum. For shoot organogenesis from leaves, the highest adventitious shoot induction rate (96.67%) with 25.67 shoots per explant was obtained when mechanically damaged leaves (the first three leaf explants at the top, leaf #1–3) were cultured with the abaxial surface placed down on Murashige and Skoog (MS) medium containing 0.5 mg L^?1 thidiazuron (TDZ). For in vitro morphogenesis of petioles, the combination of 1-naphthylacetic acid (NAA) and 6-benzylainopurine (6-BA) played a key role in cell fate determination. All of the in vitro petioles produced adventitious shoots on MS medium containing 1.0 mg L^?1 6-BA and 0.1 mg L^?1 NAA, while they produced green calli on medium fortified with 0.5 mg L^?1 6-BA and 1.0 mg L^?1 NAA. The shoots were subcultured in medium fortified with 0.5 mg L^?1 6-BA and 0.1 mg L^?1 NAA for multiplication and elongation. The elongated shoots successfully rooted on half-strength MS (1/2 MS) medium fortified with 0.5 mg L^?1 indole-butyric acid (IBA) and 0.25 mg L^?1 indole-3-acetic acid (IAA), and the regenerated plantlets successfully acclimatized with a survival rate of 92.56% in the greenhouse. The genetic fidelity of in vitro regenerated plants was evaluated using inter simple sequence repeat molecular markers. The in vitro regenerated plants were found to be the true to their mother plant. This study will be beneficial for the large-scale propagation as well as the genetic improvement of S. sebiferum.

     

Direct shoot regeneration from immature inflorescence cultures of <Emphasis Type="Italic">Chlorophytum arundinaceum</Emphasis> and <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L⁻¹ BA, 150 mg L⁻¹ Ads, 0.1 mg L⁻¹ NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation. Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L⁻¹ IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement prograrmme.     

油松成熟胚培养直接器官发生与植株再生

以油松（Pinus tabulaeformis Carr.）成熟合子胚为外植体进行了直接器官发生研究。在添加1~5 mg·L^-1 BA,或再配合添加1～10 mg·L^-1 2,4-D的DCR和MS培养基上均能诱导发生不定芽。在只添加1 mg·L^-1 BA的DCR培养基上不定芽诱导率最高,为90%。及时将不定芽继代于不含生长调节物质的DCR培养基上有利于芽的伸长。添加1 mg·L^-1 IBA的1/2DCR培养基最适于不定根的诱导,在该培养基上不定根诱导率达到47.1%。再生植株在蛭石基质上炼苗后转到温室培养,成活率高达86.4%,且生长正常。     

Micropropagation of <Emphasis Type="Italic">Cotoneaster wilsonii</Emphasis> Nakai—a rare endemic ornamental plant

A simple and efficient micropropagation system was developed for Cotoneaster wilsonii through node and shoot tip explants obtained from mature field-grown plants. Of the two explants, node explants were found to be the most effective for axillary shoot proliferation. The highest frequency of shoot induction was achieved when nodal explants were incubated on Murashige and Skoog (MS) medium supplemented with 0.5 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ α- naphthaleneacetic acid (NAA) with an average of 34 shoots per explant. The microshoots were separated from the multiple shoots and subcultured on MS medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar for further shoot growth. Maximum rooting was obtained on half-strength MS medium supplemented with 0.5 mg L⁻¹ indole-3-butyric acid (IBA). The in vitro-grown plantlets were successfully acclimatized in a glasshouse with 98% of survival. High concentrations of TDZ (1.5–2.0 mg L⁻¹) and repeated subcultures resulted hyperhydric shoots. Supplementation of the culture medium with silicon significantly reduced the induction of hyperhydric shoots. Increasing silicon concentration significantly decreased malondialdehyde content of the regenerated shoots. Data indicate that addition of silicon to the culture medium can effectively control hyperhydricity.     

Micropropagation and plant regeneration from embryogenic callus of Miscanthus sinensis

Miscanthus sinensis (Poaceae) is a typical perennial giant grass of East Asia. Due to its high photosynthetic efficiency, low input requirements, and high biomass production, M. sinensis shows outstanding potential as a biofuel feedstock. However, the lack of an efficient tissue culture system may limit its utilization potential. Different explants of M. sinensis were evaluated to develop an efficient tissue culture system. Shoot apices from in vitro-germinated seedling explants were tested for adventitious bud proliferation. The highest level of proliferation (multiplication coefficient 6.69) was obtained when shoot apices were cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ 6-benzyladenine (BA), 2.0 mg L⁻¹ kinetin, 0.05 mg L⁻¹ α-naphthalene acetic acid (NAA), 3% sucrose, and 0.8% agar. The highest rooting percentage (95.4%) was obtained when adventitious buds were cultured on half-strength MS medium supplemented with 0.2 mg L⁻¹ NAA, 3% sucrose, and 0.8% agar. Significant differences were found in the formation of embryogenic callus among different explant types. The embryogenic callus derived from epicotyls had the highest regeneration capacity when cultured on a medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid, 0.5 mg L⁻¹ BA, and 0.1 mg L⁻¹ thiamine. Under these conditions, the callus induction percentage was 82%.     

A combined pathway of somatic embryogenesis and organogenesis to regenerate radiata pine plants

This study describes for the first time in Pinus genus a plant regeneration system via a combined pathway of somatic embryogenesis and organogenesis from immature seeds of radiata pine. Somatic embryos were obtained from embryogenic line 2162 of Pinus radiata D. Don on EDM basal medium containing 60 μM ABA and 6% sucrose. The explants used for organogenesis experiments were either freshly collected somatic embryos or somatic embryos germinated for 1 week. Germination medium was half-strength LP medium, supplemented with 0.2% activated charcoal. Different induction periods and BA concentrations were assayed for shoot induction. After induction treatments, explants were elongated on the same medium used for germination stage. Rooting medium was quarter-strength LP medium supplemented with three different auxin treatments: 1.5 mg L⁻¹ 1-naphthalene acetic acid (NAA), 1.5 mg L⁻¹ indole-3-butyric acid (IBA) and 1 mg L⁻¹ IBA with 0.5 mg L⁻¹ NAA (MIX). The effect of the photon flux (120 mmol m⁻² s⁻¹ and darkness) in the first week of the explants in the rooting media was also tested. This methodology could offer an alternative to overcome some problems associated with somatic embryogenesis such as the seasonality of embryogenic tissue (ET) initiation or a low embryo production from the ET, a particularly important issue in the case of genetically transformed ETs.     

In vitro propagation of Leucocroton havanensis Borhidi (Euphorbiaceae): A rare serpentine-endemic species of Cuba

Leucocroton havanensis Borhidi is an endemic plant species of Cuba able to hyperaccumulate nickel. In order to sustain the conservation of this species, an efficient protocol for its micropropagation, via axillary bud proliferation, is described. We placed apical segments from aseptic seedlings on basal medium supplemented with indole-3-acetic acid (IAA) and 6-benzylaminopurine (BAP) or thidiazuron (TDZ), individually or in combination. On a medium containing 0.5 mg L^?1 IAA and 1.0 mg L^?1 BAP explants (65.5%) developed axillary buds. Nevertheless, combinations of 0.5 mg L^?1 IAA with 0.1 mg L^?1 TDZ was the most effective treatment producing the highest number of buds per explant (30.3); while the control treatment, without growth regulators, produced no buds at all. Transfer of buds to medium supplemented with indole-3-butyric acid, indicated that 0.25 mg L^?1 is the amount of hormone required to generate roots on young buds (100%). In order to assess DNA variations in micropropagated plants, an Amplified Fragment Length Polymorphism analysis was performed and no genetic variation was detected. This study demonstrates that a high multiplication rate can be obtained by means of the reported protocol, and that plantlets can be readily hardened (96% survival) in a greenhouse by transplanting them on serpentine soil.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

The effect of carbon source on callus induction and regeneration ability in Pharbitis nil

Flower buds, cotyledons and hypocotyls of Pharbitis nil were used as plant material. Flower buds (1–2 mm long) were excised from 3-week-old plants, grown in soil. Cotyledons of 7-day-old sterile seedlings were cut into 25 mm² squares cotyledons whereas hypocotyls were cut to 1 mm long fragments. Explants were transferred into Petri dishes containing the Murashige and Skoog medium (MS), supplemented with either BA (11 μM·L⁻¹) alone or BA (22 μM·L⁻¹) and NAA (0.55 μM·L⁻¹), and different sugars: sucrose, fructose, glucose, mannose or sorbitol (autoclaved or filter-sterilized). Addition of glucose instead of sucrose to the medium stimulated the induction of callus on flower buds and cotyledonary explants, but inhibited its growth on fragments of hypocotyls. The medium supplemented with fructose (especially filter-sterilized) stimulated the development of flower elements. Organogenesis of shoots and roots on explants was also observed. Flower buds and hypocotyls were able to regenerate both organs. Addition of fructose or glucose to the medium stimulated the organogenesis of shoots, whereas root organogenesis was inhibited on all explants used. Sorbitol strongly inhibited both induction of callus and organogenesis on all explants used.     

Direct shoot organogenesis and assessment of genetic stability in regenerants of <Emphasis Type="Italic">Solanum aculeatissimum</Emphasis> Jacq.

A simple and efficient procedure was developed for in vitro propagation of Solanum aculeatissimum Jacq. using leaf and petiole explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). Effects of various plant growth regulators, explant types, carbohydrates, and basal salts on induction of adventitious shoots were also studied. Leaf explants appeared to have better regeneration capacity than petiole explants in the tested media. The highest regeneration frequency (79.33 ± 3.60%) and shoot number (11.33 ± 2.21 shoots per explant) were obtained in leaf explants in MS medium containing 3% sucrose and 0.8% agar, supplemented with 0.1 mg/l NAA and 2.0 mg/l BA, whereas petiole explants were more responsive to 0.1 mg/l NAA and 1.0 mg/l thiadiazuron. Developed shoots rooted best on MS medium with 1.0 mg/l indole acetic acid (IAA), producing 18.33 ± 2.51 roots per shoot. Histological investigation showed that the shoot buds originated mainly from epidermal cells of wounded tissues, without callus formation. The regenerated plantlets were successfully acclimatized in a greenhouse, where over 90% developed into morphologically normal and fertile plants. Results of flow cytometry analysis on S. aculeatissimum indicated no variation in the ploidy levels of plants regenerated via direct shoot formation and showed almost the same phenotype as that of mother plants. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies of S. aculeatissimum.     

Encapsulation of cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var <Emphasis Type="Italic">botrytis</Emphasis>) microshoots as artificial seeds and their conversion and growth in commercial substrates

An effective protocol for the mass production of cauliflower microshoots was refined using the meristematic layer of cauliflower curd. After the meristematic layer was surface sterilized and shaved off, a commercial blender was used for homogenization and several blending treatments were tested in the range 15–120 s and 30 s was found to be optimal in terms of the amount explants produced and their subsequent growth ability. Explants were cultivated in S23 liquid medium (4.4 g L⁻¹ MS (Murashige and Skoog) and 3% v/w sucrose) supplemented with several combinations of plant growth regulators (PGRs) including 1 and 2 mg L⁻¹ of Kinetin in combination with three types of auxins (indole butyric acid (IBA), Naphthaleneacetic acid (NAA) and Indole-3-acetic acid (IAA)), each at 1 and 2 mg L⁻¹ concentration. The use of 2 mg L⁻¹ Kinetin and 1 mg L⁻¹ IBA gave the best results in terms of its effects on explant induction. Microshoots of different sizes were encapsulated in a sodium alginate matrix and the optimal stage suitable for the production of artificial seeds was assessed in terms of both subsequent conversion and plantlet viability. The feasibility of cultivating cauliflower artificial seeds in commercial substrates (compost, vermiculite, perlite and sand) irrigated with different solution mixtures including sterilized distilled water (SDW), PGRs-free S23 medium and S23 medium supplemented with Kinetin (1 and 2 mg L⁻¹) and IBA or NAA at (1 and 2 mg L⁻¹) was investigated. The use of 2 mg L⁻¹ Kinetin and 2 mg L⁻¹ NAA applied with S23 gave the optimal response with both perlite and compost. This study showed high growth capacity of cauliflower artificial seeds in commercial substrates which is considered a promising step for their direct use in vivo.     

Somatic embryogenic tissue establishment from mature Pinus nigra Arn. ssp. salzmannii embryos

Summary Somatic embryogenesis from mature zygotic embryos of salgare?o pine (Pinus nigra Arn. ssp. salzmannii) was induced after 2 wk of culture in L₁ medium [Murashige and Skoog mineral solution with 10.7 μM naphthaleneacetic acid (NAA) and 8.8 μM 6-benzyladenine (BA)] or L₂ medium [Gupta and Durzan mineral solution (DCR)] supplemented with the phytohormone combination as above. Four different combinations of growth regulators, NAA (2.6–10.7 μM) and BA (6.6–8.8 μM), were tried for subsequent passage. The best percentage of embryo induction and manifestation was obtained on DCR medium with 2.6 μM NAA and 6.6–8.8 μM BA. Transfer of isolated somatic embryos from the basal media to L₃ medium (Quoirin and Le Poivre modified mineral solution without hormones and supplemented with 0.3% activated charcoal), facilitated random embryo maturation and some development into plantlets.