Effect of synthetic auxins on fruit development of ‘Bing’ cherry (Prunus avium L.)

The main cherry cultivar grown in the warm climate of Israel, ‘Bing’, produces relatively small fruit. Over three consecutive years (2003–2005), application of 50 mg l⁻¹ 2,4-dichlorophenoxypropionic acid [2,4-DP; as its butoxyethyl ester (Power™)], 10 mg l⁻¹ 3,5,6-trichloro-2-pyridyloxyacetic acid [3,5,6-TPA; as the free acid (Maxim^®)], or 25 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) plus 30 mg l⁻¹ naphthaleneacetic acid (NAA; 0.3% Amigo™), at the beginning of pit-hardening when fruitlet diameter was ca. 13 mm caused appreciable and significant increases in fruit size and total yield, except when the crop load was heavy. Anatomical studies revealed that the main effect of these synthetic auxins was via direct stimulation of fruit cell enlargement. The above auxins had no negative effect on fruit quality, either at harvest or after 1 month of storage at 0 °C, or on return yield in the following year.     

Response of evergreen perennial tree crops to gibberellic acid is crop load-dependent. I: GA3 increases the yield of commercially valuable ‘Nules’ Clementine Mandarin fruit only in the off-crop year of an alternate bearing orchard

Worldwide, gibberellic acid (GA₃) is used routinely to increase fruit number and size of seedless mandarins. The efficacy of seven combinations of GA₃ concentrations and application times to maximize total yield and yield of commercially valuable fruit (diameter 57.2–76.2 mm) of ‘Nules’ Clementine mandarin (Citrus reticulata Blanco) was determined in a commercial orchard. GA₃ applied during the period of intense flower abscission failed to reduce the total number of abscised flowers in both the light off- and heavy on-bloom years. No GA₃ treatment reduced fruit abscission when trees were setting the low yield off-crop. However, all trees receiving GA₃ in the high yield on-crop year had fewer abscised fruit than untreated control trees (P = 0.0188) and GA₃ applied 10 days after 75% petal fall and in July increased the number of fruit retained on tagged branches >20% compared to control trees (P = 0.0005). Maximum air temperature was not related to flower or fruit abscission. In the off-crop year (548 fruit per untreated control tree), it was necessary to apply 15 or 25 mg L⁻¹ GA₃ at 60% bloom, 90% bloom, 75% petal fall and 10 days after 75% petal fall to significantly increase the number of fruit per tree and yield of commercially valuable fruit (kilograms and number per tree) (P < 0.0001) above that of control trees, with no reduction in total kilograms per tree. In the following on-crop year, it was better not to apply GA₃: no treatment increased total yield or fruit size and five of seven GA₃ treatments tested reduced total yield as kilograms and number of fruit per tree (P = 0.0003). The results provide strong evidence that GA₃ efficacy is crop load-dependent and dictate that crop load should be considered when using GA₃ to increase fruit set or fruit size of mandarins.     

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.     

Effect of synthetic auxins on fruit size of five cultivars of Japanese plum (Prunus salicina Lindl.)

Most of the Japanese plum (Prunus salicina) cultivars grown in Israel produce relatively small fruit. Application of 2 l solution tree⁻¹ of 25 mg l⁻¹ 2,4-dichlorophenoxypropionic acid (2,4-DP) as butoxyethyl ester (Power™), 15 mg l⁻¹ 3,5,6-trichloro-2-pyridyloxyacetic acid (3,5,6-TPA) as free acid (Maxim^®), or 25 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) + 30 mg l⁻¹ naphthaleneacetic acid (NAA) (0.3% Amigo™) at the beginning of pit-hardening, when fruitlet diameter was ca. 22 mm, caused an appreciable and significant increase in fruit size. The yield of large fruit per cv.: ‘Kesselmen’ (100% increase), ‘Songold’ (100%), ‘Black Diamond’ (800%), ‘Royal Diamond’ (160%) and ‘Royal Zee’ (100%). As a result, the total yield of all five cultivars was also increased dramatically. Anatomical studies with ‘Songold’ revealed that the main effect of these synthetic auxins was via direct stimulation of fruit cell enlargement. The above auxins had no negative effect either on fruit quality at harvest (and after 1 week in shelf-life), or on return yield in the following year.     

Parthenocarpic fruit production in loquat (Eriobotrya japonica Lindl.) by using gibberellic acid

This study evaluates the effect of gibberellic acid (GA₃) in inducing parthenocarpy in ‘Algerie’ loquat, as well as the optimum treatment conditions and associated techniques, hand thinning and ringing, to produce seedless fruit with high enough quality for fresh consumption. GA₃ applied in the course of the phenological growth stages 504–508 of the BBCH-scale produced seedless fruits, with the magnitude of the response depending on the concentration applied and number of treatments. Percentage of panicles bearing seedless fruitlets significantly increased with increasing GA₃ concentrations up to 100 mg l⁻¹ and significantly and positively correlated with the number of treatments applied. Trees treated three times with 100 mg l⁻¹ developed more than 90% of panicles bearing almost 7 seedless fruits per panicle, which were smaller in size, drier and slightly acid but similar in TSS concentration and skin colour than seeded fruits from untreated trees. Fruit thinning to 3 fruits per panicle did not increase seedless fruit size, but ringing performed at the onset of cell enlargement stage, growth stage 702 of the BBCH-scale, significantly increased fruit size by 12–15%, depending on the year. Trees treated three times with 100 mg l⁻¹ of GA₃ and ringed produced 26 kg, on average, of seeded fruit of suitable commercial quality.     

Improving ‘Bing’ sweet cherry fruit quality with plant growth regulators

Final fruit diameter is the prime determinant of sweet cherry fruit value. Previous research has shown that mesocarp cell size accounts predominantly for variability in final fruit size, within a genotype. Our research program evaluated the potential to improve sweet cherry fruit size/weight with growth regulators to affect cell division and/or cell expansion stages. In the current study we screened 8 plant growth regulators (PGRs), including cytokinins, gibberellins, and auxins, and their combinations for their ability to increase ‘Bing’ fruit weight. Each PGR was mixed in lanolin paste and applied to fruit pedicels at 9 or 30 days after full bloom (DAFB), to coincide with estimated peak in cell division and cell expansion activity, respectively. Several cytokinins applied 30 DAFB improved fruit weight significantly (ca. +15%) with N-(2-Chloro-4-pyridyl)-N′-phenylurea (CPPU) and 6-(3-hydroxybenzylamino) purine (mt-Topolin) at 100 mg l⁻¹ being the most effective. Gibberellins, applied alone, improved fruit size and delayed fruit maturation and exocarp coloration. GA₃ at 200 mg l⁻¹ applied at 9 DAFB was the most effective and improved final fruit weight by 15%. Fifty-six percent of the fruit from this treatment were ≥9 g compared to 15% of similar weight fruit from untreated limbs. Both GA₃ and GA_4/7 treatments applied 9 DAFB increased fruit radial expansion. 4-Chlorophenoxyacetic acid, a synthetic auxin, also stimulated higher fruit growth rates at stage I and stage II, and fruit color development, but did not improve final fruit size.     

Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

Clonal propagation of Rosa clinophylla Thory. through axillary bud culture

A protocol for clonal propagation of Rosa clinophylla, a rare and endangered species but very important for breeding purposes had been standardized through in vitro axillary bud culture. Although cytokinins alone were able to induce shoot buds, but their proper growth and number could be increased only when they were used in combination with GA₃. However, there was shoot tip necrosis and leaf fall in the proliferated shoots. AgNO₃ at 58.85 μM proved effective to avoid shoot necrosis and yellowing of leaves. Activated charcoal (AC) at 250 mg l⁻¹ was found necessary at all the stages of shoot multiplication as well as rooting. Ninety percent rooting could be achieved in 1/2 MS medium supplemented with 4.92 μM IBA and 250 mg l⁻¹ AC. Rooted plantlets were hardened and transferred to the field successfully with 80% survival rate.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

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.     

Molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using RAPD markers

Molecular characterization using RAPD analysis was carried out in eight cut flowers and two pot plant cultivars of chrysanthemum. Three of them (‘Refocus’, ‘Red Reagan’, and ‘Sheena Select’) were established in vitro and the occurrence of somaclonal variation was studied using the same molecular technique. Two induction media (MS + 0.1 mg l⁻¹ NAA + 0.1 mg l⁻¹ BA, and MS + 2.0 mg l⁻¹ IAA + 0.5 mg l⁻¹ Kinetin), and two proliferation media (MS + 0.1 mg l⁻¹ NAA + 0.2 mg l⁻¹ BA, and MS + 4.0 mg l⁻¹ IAA + 2.0 mg l⁻¹ Kinetin) were employed in order to evaluate the effect of the medium composition in the shoots’ stability. Likewise, the effect of the culture age was considered in assessing genetic stability. Monthly subcultures were carried out, identifying the origin and history of the shoots, throughout a nine-month proliferation period followed by acclimatization. Molecular markers were obtained in every subculture cycle and from the acclimatized plants. Only one shoot from the 7th subculture of the cultivar ‘Refocus’ showed a different band pattern. The use of RAPD for chrysanthemum cultivar characterization and somaclonal variation detection is discussed.     

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.     

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.     

Effects of high electrical conductivity of nutrient solution and its application timing on lycopene,chlorophyll and sugar concentrations of hydroponic tomatoes during ripening

Tomato (cv. Durinta) plants were grown hydroponically under two electrical conductivities (EC, 2.3 and 4.5 dS m⁻¹) of nutrient solution inside a greenhouse. The high EC treatment was initiated either immediately after anthesis (high EC treatment) or 4 weeks after anthesis (delayed high EC treatment). Fruits were harvested weekly beginning 2 weeks after anthesis, until all fruits reached the red stage (8 weeks after anthesis). Lycopene, chlorophyll, sugar and total soluble solid (TSS) concentrations of fruits were measured every week for all harvested tomatoes from the different ripeness stages. The results showed that lycopene concentration, fructose and glucose concentrations and TSS of red ripe tomato fruits were enhanced by both high EC and delayed high EC treatments compared to those in the low EC treatment. The lycopene concentration of red ripe tomato fruits in the high EC and the delayed high EC treatments showed an increase of 30–40% (1.29–1.39 mg g⁻¹ dry matter) compared to those in the low EC treatment (0.99 mg g⁻¹); however, there was no significant difference in the lycopene concentration between the high EC and delayed high EC treatments. TSS of red ripe tomato fruits grown in the high EC treatment was 6.1%, significantly greater than those grown in the delayed high EC treatment (5.7%). Weekly change in lycopene concentration indicated that lycopene synthesis was enhanced by the high EC treatment, regardless of the application timing. Regardless of EC treatment, chlorophyll concentration in fruit declined linearly during fruit development and ripening and reached non-detectable levels 7 weeks after anthesis. Our results indicated that: (1) accumulation of sugars and TSS in fruit was due to reduced water flux to the fruit under high EC as previously reported, and (2) lycopene synthesis was promoted by, but chlorophyll degradation was independent from, the osmotic and/or salt stress caused by the high EC.     

Factors affecting haploid induction through in vitro gynogenesis in summer squash (Cucurbita pepo L.)

Haploid production using in vitro ovule cultures has long been recognized as an important tool to produce haploid and homozygous double-haploid plants for genetic studies and plant breeding programs. In the present study, four experiments were carried out to study the influence of genotype, position of female flowers on plant stem, temperature and sucrose concentration on the in vitro gynogenesis induction of squash. (1) Ovules of 12 genotypes were excised from female flowers, 1 day before anthesis, and cultured onto MS medium containing 3% sucrose and 1 mg l⁻¹ from each of kinetin and 2,4-D (2,4- dichlorophenoxy acetic acid). Differences in response among genotypes were demonstrated. Raad F₁ showed the highest percentage of responding ovules and number of plantlets per dish with 48.8% and 15 plants, respectively. The results revealed that genotype is a key factor influencing the in vitro gynogenesis in squash. (2) Ovules were excised from first, second and third female flower of two hybrids (Giad and Raad) and cultured onto the mentioned above medium. The highest percentage of responding ovules and number of plantlets per dish were obtained from ovules excised from the second female flower on the plant stem. (3) Effect of temperature (4 and 32 °C) for 0, 4, 7 and 12 days on the ovule culture of Queen F₁ was studied. Ovules incubated at 4 or 32 °C for 4 days produced a better embryogenic response. (4) Three sucrose concentrations (30, 60 and 90 g l⁻¹) were tested with the ovule cultures of the local cultivar (Eskandrani). Differences among sucrose concentrations were statistically significant and ovules cultured on the MS medium containing 30 g l⁻¹ produced the best result. MS medium containing 90 g l⁻¹ did not produce gynogenic ovules.     

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.     

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.     

Regeneration and transformation system in Mirabilis jalapa

Protocols for in vitro regeneration and production of in vitro-propagated plants and a transformation system were developed for Mirabilis jalapa (Nyctaginaceae). Among the types of explants and the different media tested, consistent shoot regeneration was obtained only from nodal segments grown in a regeneration medium consisting of Murshashige and Skoog medium supplemented with 2 mg l⁻¹ 6-benzyladenine, 2 mg l⁻¹ zeatin and 1 mg l⁻¹ indole acetic acid. Regeneration efficiency was dependent on the type of plant – white or pink flowers – used as the source of explants. Stable transformation was obtained following inoculation of nodal segments with Agrobacterium tumefasciens strain EHA105, which harbours the binary plasmid pAD1339 containing both nptII and gus genes under the control of the 35S promoter. Transformation was confirmed by PCR and Southern blot analysis of genomic DNA from mature regenerated plants. β-Glucuronidase (GUS) activity was observed only in tissues regenerated from in vitro-grown plants and not in tissues originating from greenhouse-grown plants. GUS expression was not uniform in regenerated leaves and showed a chimera pattern.