Development of a plant regeneration system from seed-derived calluses of centipedegrass [Eremochloa ophiuroides (Munro.) Hack]

The objective of this study is to establish plant regeneration system with the seed of the new Chinese selection “E-126”of centipedegrass [Eremochloa ophiuroides (Munro.) Hack] as explant. In present study, the following results were obtained: (1) The medium formulation most suitable for calluses induction was identified to be MS with 1.0 mg l⁻¹ 2,4-D + 30 g l⁻¹ mannitol + 50 ml l⁻¹ coconut milk and the ratio of calluses induction was 96.0%, including 5.2% of yellow granule calluses induction. The above medium formulation was adopted for subculture. (2) The rate of shoot regeneration from yellow granular calluses was 98.0% by MS optimum medium formulation with 2.0 mg l⁻¹ KT + 50 ml l⁻¹ coconut milk. The differentiated rate retained as high as 88.0% even after 5 times of subculture and 18.6% after 15 times of subculture. The optimum medium formulation for shoot growth was identified to be MS medium plus 2.0 mg l⁻¹ BAP, 0.8 mg l⁻¹ NAA and 50 ml l⁻¹ coconut milk. (3) The optimum medium for shoot rooting was identified to be MS medium with 0.6 mg l⁻¹ NAA + 50 ml l⁻¹ coconut milk, and the rooting rate to be 98.0%. The survival rate of transplanted plantlets from tubes to basin with soil was 92.0%. In conclusion, the plant regeneration system was successfully developed in this study, which may provide basic reference for screening of somaclonal variants and genetic transformation of centipedegrass.     

Direct plant regeneration from mature leaf explants of pistachio, Pistacia vera L.

A protocol was developed for direct shoot and plantlet regeneration from in vitro regenerated leaf explants of male Pistacia vera L. cv. ‘Atl?’. Leaves excised from axenic shoot cultures of pistachio were used to induce organogenesis on a Murashige and Skoog (MS) medium with Gamborg vitamins supplemented with combinations of different concentrations of BAP and IAA. The highest adventitious shoot regeneration in 35% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants cultured during the establishment phase in the medium with 1 mg l⁻¹ IAA and 2 mg l⁻¹ BAP. For shoot multiplication, the highest number of new microshoot/explants (5.76) was obtained in a culture medium supplemented with 1 mg l⁻¹ BAP, but it was not significantly different from the number obtained at 2 mg l⁻¹ BAP. A high rooting frequency (84%) for microshoots was recorded on a medium supplemented with 2 mg l⁻¹ IBA. In vitro rooted plantlets were transferred to pots filled with a mixture of soil, sand and peat (1:1:1). They were weaned in a growth room and finally moved to a greenhouse. This protocol could be utilized for in vitro clonal propagation of this economically important plant.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.     

Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.     

In vitro regeneration from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit

Methods to regenerate whole plants from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit were established. To optimize shoot induction, leaf explants were cultured on media containing different types and combinations of plant growth regulators. Growth was initiated within 17–24 days culture, and included callus formation, and root or shoot organogenesis ranging from 20 to 100% regeneration. Shoots were induced only when explants were cultivated on MS medium containing a combination of NAA and kinetin, NAA and BAP, IAA and Kinetin, or IAA and BAP. On media containing NAA and BAP, dark incubation was critical for efficient direct shoot regeneration from explants. Direct shoot formation and the highest number of shoots per explant (17.6) were obtained from leaf explants cultured in the dark for 30 days on MS medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ BAP. Shoots cultured on MS medium containing 0.1 mg l⁻¹ NAA formed tuberous roots with microtubers within 42 days. Healthy regenerated plants were acclimated and transferred to a greenhouse.     

Micropropagation of the bromeliad Guzmania ‘Hilda’ via organogenesis and the effect of α-naphthaleneacetic acid on plantlet elongation

This study established a highly effective micropropagation system to obtain good plantlet proliferation from floral organs via callus induction and bud differentiation in Guzmania ‘Hilda’ bromeliad. The best frequencies of organogenic callus formation (20% in petal and 35% in ovary explants) were obtained on media containing a combination of 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.5 mg l⁻¹ 2,4-D with 0.5 mg l⁻¹ NAA, respectively. Organogenic calli were cultured on medium with 1.0 mg l⁻¹ NAA and 0.5 mg l⁻¹ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) induce the differentiation and regeneration of adventitious buds into plantlets. When the plantlets were cultured in a medium with optimum NAA concentration (0.5–1.0 mg l⁻¹) significant improvement in regeneration and elongation was achieved within one month. This overcame the difficulty of delayed elongation in Guzmania plantlets. More than 99% of the regenerated and acclimatized plantlets developed to the flowering stage.     

Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.)

Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented with either YE or CH at 0.0, 0.1, 0.25, 0.5, and 1 g l⁻¹. To induce somatic embryogenesis, callus was transferred to a hormone-free medium containing the corresponding concentration of additives. The results have shown that callus weight and the number of somatic embryos were directly proportional to increases in the concentration of organic additives tested. Callus growth was best achieved when 1 g l⁻¹ of either YE or CH was added to the culture medium. At this concentration of YE, callus growth was double that of the control medium. On CH-containing media growth was 2.3 times that of the control. This indicates that CH is more effective in enhancing callus growth. However, YE was more effective in enhancing somatic embryogenesis. The data show that the best somatic embryo formation was obtained on either 1 g l⁻¹ YE or 0.5 g l⁻¹ CH which produced 45 and 30 embryos per culture, respectively, as compared to 20 embryos produced in the control treatment. Resultant somatic embryos successfully rooted and regenerated plantlets which exhibited normal growth in the greenhouse. Enhanced plant regeneration, an essential criterion for commercial micropropagation, was achieved.     

Priming abiotic factors for optimal hybrid Cymbidium (Orchidaceae) PLB and callus induction,plantlet formation,and their subsequent cytogenetic stability analysis

Abiotic factors affect the induction of PLBs and callus in hybrid Cymbidium Twilight Moon ‘Day Light’. The initiation and proliferation of new PLBs and callus could be achieved on NAA and kinetin, supplemented at 0.1 mg l⁻¹ each, respectively, both within 45–60 days. Bacto agar was found to be the most suitable solidifying agent for PLB induction, although a higher shoot fresh weight was obtained on Gelrite; a pH 5.3 was optimal while pH 4.5 caused 100% explant necrosis; coconut water, when supplied at 10–20% (v/v) resulted in a significant increase in the number of PLBs formed per PLB segment (23.1 versus 14.6 in controls) while a massive (almost four-fold) increase in fresh top weight occurred when PLB explants were placed in liquid culture, as a result of hyperhydricity; Fe-EDTA (1 mg l⁻¹) and activated charcoal (1 g l⁻¹) stimulated total fresh weight and PLB formation in the presence of PGRs; PLB formation decreased but total fresh shoot weight increased with the addition of niacin or myo-inositol, both vitamins. Dark-grown PLB-induced plants were etiolated and had longer internodes and higher fresh weight than light-grown control plants at 45 μmol m⁻² s⁻¹; at 15 μmol m⁻² s⁻¹ shoots were slightly etiolated, fragile, and PLB formation was scarce. RAPD and mtDNA analysis of all resultant PLBs, callus or plants showed them to be genetically identical, with comparable chlorophyll contents. Despite the detection of cytological variation between different plant parts, little variation resulted from abiotic factor treatment.     

Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus

An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l⁻¹ 2,4-d plus 8.9 μmol l⁻¹ BA, callus subculture on medium F (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 0.44 μmol l⁻¹ BA) and then on medium T (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 2.2 μmol l⁻¹ BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l⁻¹GA₃, 23.6 μmol l⁻¹ AgNO₃, 0.02% active charcoal and 4.5 μmol l⁻¹ TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l⁻¹, TDZ 9.1 μmol l⁻¹ and GA₃ 8.7 μmol l⁻¹), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l⁻¹ NAA and 1.1 μmol l⁻¹ BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.     

Embryo germination and proliferation in vitro of Juglans regia L.

This paper presents the optimal culture conditions for the in vitro embryo germination and proliferation of Juglans regia L. rootstock cv. Peralta, selected by the IMIDA fruticulture team. J. regia L. rootstock cv. Peralta is characterised by its resistance to salinity, lime and by its vigour. The first experiment determined the best culture medium for in vitro embryo germination. The Murashige and Skoog medium (MS; Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissues cultures. Physiol. Plant. 15, 473–497), the medium developed by our team for walnut (NGE), the Lloyd and McCown medium (WPM; Lloyd, G., McCown, B., 1981. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by the use of shoot tip culture. Proc. Plant Prop. Soc. 30, 421–427) and that of Driver and Kuniyuki (DKW; Driver, J.A., Kuniyuki, A.M., 1984. In vitro propagation of Paradox walnut rootstock. HortScience 19 (4), 507–509), were compared, all without growth regulators. The best germination percentage was obtained in WPM (81%) with significant differences between the different media. In the second experiment, the optimal benzylaminopurine and indole butyric acid concentrations were determined for the proliferation stage of the explants obtained in the first experiment. The proliferation rates obtained varied from 0 in the medium without cytokinins to 6 in the medium with 0.5 mg l⁻¹ BAP. The cluster proliferation quality and other parameters studied indicate that the optimal treatment was 0.5 mg l⁻¹ BAP.     

Conservation of Vanilla species, in vitro

Vanilla (Vanilla planifolia) is a crop of great commercial importance as the source of natural vanillin, a major component of flavor industry. The primary gene pool of V. planifolia is narrow and is evidently threatened due to destruction of its natural habitats making the secondary gene pool important as a source of desirable traits especially for resistance to diseases. Many species of vanilla are considered rare and endangered hence an urgent need to conserve them, arises. Effective procedures for micropropagation and in vitro conservation by slow growth in selected species of vanilla, are described. Synthetic seed technology was standardized by encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, which could be stored up to 10 months with 80% germination in sterile water at 22 ± 2 °C. In vitro conservation technology of Vanilla was standardized and shoot cultures could be maintained for more than 1 year without subculture, on slow growth medium, i.e. Murashige and Skoog medium supplemented with 15 g l⁻¹ each of sucrose and mannitol in sealed culture vessels at 22 ± 2 °C. These cultures were maintained in vitro for more than 7 years with yearly subculture. The conserved material could be retrieved and multiplied normally in MS medium with 1.0 mg l⁻¹ BA and 0.5 mgl ⁻¹ IBA. The in vitro conserved plants showed good growth and developed into normal plants. This synseed and in vitro conservation system can be utilized for conservation and exchange of vanilla genetic resources.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Effect of genotype,explant size,position, and culture medium on shoot generation of Gerbera jamesonii by receptacle transverse thin cell layer culture

An unique procedure for the mass shoot propagation of Gerbera using receptacle transverse thin cell layer (tTCL) culture procedure was developed. Genotype, flower bud age, explant size, position of receptacle tTCLs and culture media were found to affect the success of culture. Ten interspecific crosses of Gerbera showed different shoot regeneration rates and callus induction via receptacle tTCL culture, all of which had shoot regeneration rates higher than 57%. Flower buds collected on the 10th day resulted in 91% shoot regeneration after 6 weeks of culture on basal MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 475–497] supplemented with 0.02 mg l⁻¹ thidiazuron (TDZ), 0.8 mg l⁻¹ adenine and 10% (v/v) coconut water (CW). This was significantly higher than those from flower buds on the 7th and 14th days (22% and 54%), respectively. Shoot regeneration rate was the highest (94–100%) in the middle layers of the receptacle. For mass shoot propagation, shoot clusters were subcultured on half-strength MS medium supplemented with 0.5 mg l⁻¹ indole-3-butyric acid (IBA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 2.0 mg l⁻¹ kinetin after every 4 weeks. Plantlets formed when single shoots were cultured on half-strength MS medium containing 1 mg l⁻¹ IBA. All plantlets acclimatized well in the greenhouse.     

Meristem culture and micropropagation of a variety of ginger (Zingiber officinale Rosc.) with a high yield of oleoresin

Summary

Meristems of ginger with or without leaf primordia were induced to form shoots on three-quarter strength Murashige-Skoog’s (MS) medium containing sucrose 6%, coconut milk (CM) 20%, ascorbic acid (AA) 100 mg l^?1, glutamine (GL) 400 mg l^?1, activated charcoal (AC) 250 mg l^?1, 6-benzylaminopurine (BAP) 0.5 mg l^?1, indolebutyric acid (IBA) 0.4 mg l^?1 and agar 0.8%. Meristem-derived shoots exhibited consistent multiplication on three-quarter strength MS medium containing sucrose (3%), AA (100 mg l^?1), AC (100 mg l^?1), BAP (4–5 mg l^?1) and agar (0.8%). Liquid media (agitated or static) were less effective than a solid (agar-gelled) medium for micropropagation. Kinetin and naphthalene acetic acid (NAA) incorporated at various levels (0.01–0.8 mg l^?1) with or without added BAP and IBA neither improved plantlet formation nor enhanced shoot multiplication. The in vitro plants were successfully established in vivo and the rhizome yield was comparable with that of plants grown by conventional methods.     

Effect of 2,4-d, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.)

The proliferation of embryogenic suspension culture in two cultivars (Jihel and Bousthami Noir) of Phoenix dactylifera L. was tested on liquid media with or without 2,4-d and with different glutamine concentrations (3.35 × 10⁻⁴, 6.7 × 10⁻⁴ and 13.4 × 10⁻⁴ M). The liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine has clearly improved the proliferation of somatic embryos. In fact, when glutamine concentration increased from 3.35 × 10⁻⁴ to 6.7 × 10⁻⁴ M, the yield of somatic embryos increased from 14 to 56 embryos per 100 ml of culture medium for “Jihel” cultivar and 25–71 embryos per 100 ml of culture medium for “Bousthami Noir” cultivar. In contrast, increasing glutamine concentration from 6.7 × 10⁻⁴ to 13.4 × 10⁻⁴ M, the embryos yield was negligible. Based on biochemical analysis, the highest accumulation of proteins and sugars was obtained in liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine (118 and 91 mg of proteins g⁻¹ DW, respectively, for “Jihel” and “Bousthami Noir” cultivars; 194 mg of sugars g⁻¹ DW for “Jihel” cultivar and 182 mg of sugars g⁻¹ DW for “Bousthami Noir” cultivar). In addition, the supply of 0.05 mg l⁻¹ BAP on the germination medium could be useful in terms of germination percentage of somatic embryos. When BAP concentration increased from 0.05 to 0.2 mg l⁻¹, the germination percentage of somatic embryos decreased from 14.2 to 4.9%, while secondary embryogenesis increased from 26.4 to 45.2%.     

Plant regeneration from stem segment-derived friable callus of “Fonio” (Digitaria exilis (L.) Stapf.)

“Fonio” (Digitaria exilis (L.) Stapf.) is a member of the grass family with excellent culinary and nutritional properties. In spite of its economic values, hardly has any improvement work been done. To enhance genetic improvement of this grain, plant regeneration protocol was developed using 8 cultivars. Stem segments of 5 mm long excised from 1 month-old seedlings germinated in vitro were cultured on 6 types of media for friable callus induction. Best result was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 g l⁻¹ casamino acid, where 91.3, 88.9 and 87.8% of the explants formed friable calli in cultivars ‘Kurelep’, ‘Churiwe’ and ‘Agyong’, respectively. Shoots appeared when friable calli were transferred to two regeneration media, i.e., MSBZ (MS medium + 0.022 mg l⁻¹ 2,4-D, 0 .22 mg l⁻¹ 6-benzylaminopurine (BA), 0.22 mg l⁻¹ zeatin) and MSBG (MS medium + 0.5 mg l⁻¹ BA, 0.1 mg l⁻¹ gibberellic acid). The highest frequency of plant regeneration was attained on MSBG, with 91.7% of the friable calli forming shoots in cultivar “Churiwe”. Regenerated plants were rooted on hormone free MS medium. Flow cytometric analysis revealed 100% of the regenerants to be diploid. The protocol developed here can be used in the transformation of “Fonio” to increase the yield potential of this crop by incorporating characteristics such as disease resistance and stress resistance.     

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l⁻¹ BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l⁻¹ BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.     

High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO₃ on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l⁻¹, 6-BA at 3.0 mg l⁻¹, AgNO₃ at 4.0 mg l⁻¹ and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l⁻¹, respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.     

Efficient in vitro multiplication in Ornithogalum ulophyllum Hand.-Mazz. from twin scale explants

Ornithogalum ulophyllum Hand.-Mazz. with beautiful white flowers is an important medicinal and ornamental plant of the Middle Eastern countries and need exploitation for commercial propagation. The study reports in vitro mass proliferation of bulblets achieved from twin scales and “in vitro regenerated bulblet” explants on MS medium supplemented with various concentrations of BAP–NAA. The best regeneration on twin scales and “in vitro regenerated bulblets” was obtained on MS medium containing 2 mg l⁻¹ BAP–0.5 mg l⁻¹ NAA and 2 mg l⁻¹BAP–1 mg l⁻¹ NAA, respectively. However, bulb scales seemed to be more potent for bulblet regeneration. A large number of the developing bulblets rooted on the regeneration medium. Remaining non-rooting bulblets were rooted on MS medium containing 1 mg l⁻¹ NAA. All plants were acclimatized in the environmental chamber for 4 weeks and were transferred to the greenhouse for flowering. Regenerated bulblets developed into morphologically normal plants.     

Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs

A reproducible procedure was developed for genetic transformation of Hydrangea macrophylla Ser. cv. Blaumeise by Agrobacterium tumefaciens following the development of an efficient regeneration system using leaf discs excised from 12 to 15 weeks old meristem-derived vitroplants. Explants were cultivated on solid B5 medium complemented with maltose 110 mM, BAP 10 μM and NAA 0.5 μM. A low light regime of 17 μmol m⁻² s⁻¹ improved regeneration frequency up to 86%. For transformation, leaf discs were inoculated and co-cultivated with two disarmed A. tumefaciens strains, EHA 101 and LBA 4404, both carrying the binary vector pFAJ3000 which contained the nptII selectable gene and the GUS reporter gene. A pre-culture period of 3 days and a short co-cultivation duration (1 day) improved the efficiency of transformation. Inoculation of only 10 min with agitation including (or not) vacuum infiltration was sufficient. If selection on kanamycin containing medium was applied after a 2 weeks culture period on shoot regeneration medium, the percentage of explants forming kanamycin-resistant shoots increased from 3.3 to 13.3%. Integration and expression of the introduced transgene were confirmed by histochemical GUS assay, PCR and Southern blot analysis. Flowering of transgenic plants in glasshouse occurred 10 months after acclimatization.