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.     

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium guajava L.)

Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava (Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l⁻¹) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l⁻¹) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l⁻¹ ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l⁻¹) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.     

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.     

Callus induction and plant regeneration from embryonic axes of Kosteletzkya virginica

Kosteletzkya virginica, a perennial dicot halophytic species of the Malvaceae, is native to American salt marsh. It was introduced into China as a potential species to improve coastal wetlands and to develop ecologically sound saline agriculture. K. virginica adapts excellently to the tidal-flat habitats in China's east coast, with multiple eco-benefits; in particular, its seed oil could be used to produce biodiesel. The purpose of this study was thus to develop a standardized protocol to induce a high frequency of callus and subsequent plantlet regeneration system for a K. virginica breeding program with the final objective of applying transgenic techniques to improve seed oil yield. The embryonic axes of K. virginica were used as explants for callus induction, shoot induction from the callus and then adventitious root induction from the shoots on nine culture media with different hormone combinations. The best results were achieved on the following media: (1) 93.94% callus induction on MS medium supplemented with 1.0 mg L⁻¹ indole-3-acetic acid (IAA), 0.3 mg L⁻¹ kinetin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (2) 65.83% shoot induction on 1/2MS medium supplemented with 0.1 mg L⁻¹ IAA, 0.5 mg L⁻¹ zeatin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (3) 96.67% rooting on MS medium containing 30 g L⁻¹ sucrose and 8 g L⁻¹ agar. The survival rate of plantlets by organogenic regeneration was 85% after being transplanted into potting soil in flowerpots and placed in the greenhouse. This experiment indicates that we established successful callus induction and plant regeneration protocols for K. virginica.     

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.     

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.     

High frequency embryogenic callus induction and plant regeneration from mature caryposis of big bluestem and little bluestem

Big bluestem (Andropogon gerardii Vitman) and little bluestem [Schizachyrium scoparium (Michaux) Nash.] are native to the North America and are important forage grasses and ornamental grasses. Both grasses are proposed as ideal biomass producers for cellulosic ethanol production. To apply genetic transformation, which is an important tool for incorporating desirable agronomic traits into plants to both species, however requires an efficient and reproducible regeneration protocol. We used mature caryopses from big and little bluestem as explants and tested the effect of various combinations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) (1, 2, 3, 4 or 5 mg l⁻¹) and kinetin (KT) (0, 0.1 or 0.2 mg l⁻¹) on embryogenic callus induction with LS as the basal medium. The highest percentage of embryogenic calli induction occurred on medium containing 2, 4-D alone at 2 mg l⁻¹ for ‘Bison’ and on medium containing 4 mg l⁻¹ 2, 4-D alone for ‘Bonilla’ big bluestem. For little bluestem, the highest percentage of embryogenic callus induction occurred on medium containing 3 mg l⁻¹ 2, 4-D plus 0.1 mg l⁻¹ kinetin, suggesting that addition of KT is beneficial. Shoot regeneration took place on LS basal medium without any plant growth regulator for both species, although the addition of KT increased both regeneration frequency and the number of shoots produced per callus. Rooting of shoots reaching about 2 cm long occurred readily with or without α-naphthaleneacetic acid (NAA). Rooted plantlets were all successfully established in the soil.     

High frequency early flowering from in vitro seedlings of Dendrobium nobile

Dendrobium nobile Lindl. is a popular temperate Chinese orchid commonly marketed as a traditional medicinal plant. Seedlings of Dendrobium nobile Lindl. produced floral buds (33.3–34.8%) precociously on a defined basal medium (1/2 MS) containing paclobutrazol (PP₃₃₃) at 0.5 mg L⁻¹ or thidiazuron (TDZ) at 0.1 mg L⁻¹ within 4 months of culturing. The frequency of floral buds formation can be further increased to 95.6% by growing seedlings in a PN (PP₃₃₃ 0.3 mg L⁻¹ + NAA 0.5 mg L⁻¹)-containing medium followed by transfer onto 1/2 MS medium with PP₃₃₃ and TDZ (PP₃₃₃ + TDZ). However, flower developed was deformed under 25 °C but it developed fully when grown in a lower temperature regime (23 °C/18 °C, light/dark) for 45 days. Under optimal condition, in vitro flowering was observed about 6 months after seed sowing.     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

Spectral effects on embryogenesis and plantlet growth of Oncidium ‘Gower Ramsey’

Embryogenic calli of Oncidium were grown under tubular fluorescent lamp (FL) or light-emitting diode (LED) arrays with different spectra to determine the effects of light quality on protocorm-like body (PLB) formation and plantlet growth from the callus. The effect of light is generated by monochromatic blue, red or mixed far-red LEDs on the photomixotrophic radiation. The light sources induced higher number of PLB formation compared with the darkness. The best condition for PLB formation was to culture on MS half salt medium supplemented with 0.1 mg l⁻¹ NAA and 0.4 mg l⁻¹ BA under red + blue + far-red (RBFr) LEDs and FL radiation for 8 weeks. Roughly 3000 embryos were induced in an initial culture of 1 g of fresh weight of calluses. During subculturing, PLBs had the ability to convert into plantlets. In this study also indicated the far-red LED radiation was a noticeable factor. It showed that using Fr combined with R and B (RBFr) or RFr radiation significantly enhanced leaf expansion, numbers of leave and root, chl contents, fresh and dry weight of Oncidium plantlets. Therefore, the wavelength specificity of RBFr LEDs comprising a novel illumination system is advantageous over FLs for PLB formation, resulting in higher efficiencies of plant regeneration and growth in Oncidium cultures.     

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.     

Recurrent somatic embryogenesis and plant regeneration in Coriandrum sativum L.

An efficient method of repetitive somatic embryogenesis and plant regeneration was established in Coriandrum sativum L. Embryogenic callus was induced from cotyledon and hypocotyl segments on Murashige and Skoog (MS) medium with 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), upon subculturing on medium having same level of 2,4-D at an interval of 3 weeks developed somatic embryos, which progressed to cotyledonary stage through early developmental stages of somatic embryogenesis. The transfer of somatic embryos at an early cotyledonary and cotyledonary stage in clumps in succession to fresh 4.52 μM 2,4-D supplemented medium developed embryos in a cyclic manner. Upon transferal to embryogenic clumps (cotyledonary embryos) to modified MS medium (4 g l⁻¹ KNO₃, 0.29 g l⁻¹ NH₄NO₃, 3 mg l⁻¹ thiamine HCl, 0.5 mg l⁻¹ pyridoxine HCl, and 5 mg l⁻¹ nicotinic acid), the embryos irrespective of the cycles underwent maturation and germination. Germinating embryos transferred to half-strength MS medium favored healthy growth of plantlets. The system of recurrent somatic embryogenesis in coriander offers a system for genes transfer and also scale-up production of modified plants.     

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.     

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%.     

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.     

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.     

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.     

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.     

High frequency in vitro embryogenic callus induction and plant regeneration from indiangrass mature caryposis

Indiangrass [Sorghastrum nutans (L.) Nash.] is native to the North America and is an important component of the original tall grass prairie. It is also an important ornamental and forage grass. Recently, it has been proposed as an ideal biomass producer for cellulosic ethanol production. Genetic transformation is an important tool for introducing important agronomic traits into plants, but an efficient and reproducible in vitro regeneration protocol is a prerequisite for successful genetic transformation. In this report, we used mature caryopses as explants and tested the effect of various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (1–5) and kinetin (KT) (0, 0.1, and 0.2) on embryogenic callus induction using LS basal medium. Caryopses cultured on media supplemented with 2,4-D alone generally outperformed those cultured on media supplemented with both 2,4-D and kinetin for embryogenic callus induction. The best treatment is LS basal medium supplemented with 3 mg l⁻¹ 2,4-D. LS basal medium supplemented with KT of 0, 0.5, 1, 2 or 5 mg l⁻¹ were tested for regeneration efficiency which was shown to increase as the KT concentration increased. The quality of the shoots produced on the medium containing KT at 5 mg l⁻¹, which produced the highest regeneration frequency appeared to be lower as leaves become vitrified. Shoots were moved to a rooting medium containing either 0 or 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA). Rooted plantlets were then transferred to soil-containing pots and were placed in a mist room for 1 week before they are transferred to a normal greenhouse where they all survived. The reported regeneration protocol is very efficient and highly reproducible in spite of the heterogeneous nature of the tested cultivar; thus it should be suitable for genetic transformation.     

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.