Eggplants transformed with Sw-5 gene, regenerated by organogenesis and somatic embryogenesis, were resistant to the Tomato chlorotic spot virus, while wild plants did present systemic infection. TO plants were selfed and the segregation analysis of T1 and T2 generation indicated the existence of one or more i...nsertion sites. Southern blot analysis confirmed one or two independent insertions in T2 plants. Different lesions associated with the insertion number were observed in TI and T2 plants. T2 plants with two copies displayed faster hypersensitive reactions and characteristic necrotic lesions that contrasted with slower responses and necrotic ring lesions in plants with one copy. These results suggest that the Sw-5 confers resistance to tospovirus in transgenic eggplants and that the resistant phenotype depends on the number of transgene copies.
The effects of kanamycin and hygromycin-based selection and co-culture temperature ranging from 22 to 28 °C upon eggplant transformation efficiency were evaluated. Both morphogenic pathways, somatic embryogenesis and organogenesis, were adopted using cotiledonary and hypocotyl explants, respectively. Somatic embryos were recovered in the prese...nce of both antibiotics, although lesser escapes were observed in hygromycin-supplemented medium. Indeed, selection provided by this antibiotic was more efficient compared to kanamycin, nevertheless, shoot regeneration was not observed with hygromycin. Significant difference on the frequency of cotiledonary explants displaying callus (FEC) was observed as embryogenesis was concerned, although a higher number of embryos was observed in hygromycin selective media. The frequency of explants presenting callus (FEC), embryos (FEE) and shoots or buds (FERG) did not differ statistically for the tested co-culture temperatures, although higher regenerant number was observed at 24 °C.
The influence of antibiotics (cefotaxime, timentin, kanamycin and hygromycin) and growth regulators (indolacetic acid and 6-benzylaminopurine) was evaluated on eggplant organogenesis. Solanum melongena hypocotyl segments (6 to 10 mm length), taken from 16 to 20-days in vitro grown seedlings, were used as explants. The basic medium was...composed by MS salts, Gamborg vitamins and 2% sucrose, solidified with agar 0.8% and pH adjusted to 5.7±0.2. Morphogenesis was impaired at 50 to 100 mg L-I kanamycin and 7.5 mg L-1 hygromycin. Both Timentin and cefotaxime reduced the frequency of regenerating explants meanwhile hyperhydricity was not affected. A decrease in root regeneration was observed with increasing cefotaxime concentrations, although, timentin had no effect on root regeneration, as compared to the control treatment. Interestingly, the number of adventitious roots was more noticeable at 0.25 mg L-I IAA plus 0.5 mg L-1 BAP. However, if just IAA was added led to higher number of regenerated roots compared to other treatments.
The effects of the ethylene precursor ACC and two inhibitors, AgNO3 and AVG, on root formation were tested in in vitro shoots of passion fruit (Passiflora Midis f.flavicalpa Deg.). The organogenic response was assessed on the basis of percentage of shoot-forming. roots, root number and length. The time course of eth...ylene production was also monitored. ACC inhibited root formation by delaying root emergence and increasine, callus formation at the basis of the shoots. In addition, ACC caused a marked increase in ethylene production, coupled to leaf chlorosis and senescence with lower rooting frequencies, number and length of roots. IAA supplementation increased ethylene production. Both ethylene inhibitors, AgNO3 and AVG, at appropriate concentrations reduced callus formation at the basis of shoots. AVG increased the number of roots per shoot, but drastically reduced length of differentiated roots. Regarding to leaf pigments, ACC promoted a marked reduction on carotenoids and total chlorophyll, whereas AVG and AgNO3 delayed explant senescence and pigments degradation, not differing from IAA supplemented and non-supplemented control treatments. The results confirm previous reports on the beneficial effects of ethylene inhibitors on in vitro rooting and suggest its reliability to be used as an alternative approach to evaluate sensitivity of Passiflora species to ethylene.
Hyperhydricity was observed throughout in vitro multiplication phase of a Eucalyptus grandis clone. Ultrastructural approach of tissue and cell differentiation, izoenzyme patterns, binding protein (BiP) expression, and pigment content were performed. Hyperhydric tissues showed a reduction in cell wall deposition, reduction of...membranous organelles, higher cell vacuolation, and more intercellular spaces than its normal counterpart. Additionally, several vesicles were present in hyperhydric cells suggesting the occurrence of organelle autophagy by autophagic vacuole. Lower pigment content, intercellular spaces on the epidermis and the induction of a molecular chaperone (BiP) were observed in hyperhydric phenotype. Evidences of schizolysigenous process of intercellular space formation are compatible with a stress condition. Although plastoglobulli were observed in normal and hyperhydric chloroplasts, they were more evident in the normal ones. Abnormal stomata also reflected a disruptive situation and morphogenesis disturbances which would difficult plant acclimatization. Further observation of the epidermis ultrastructure allows us to conclude that the presence of intercellular spaces on its surface may be constraining the recovery and development of hyperhydric plants. Similarly to BiP, other proteins such as esterase (EST), acid phosphatase (ACP), malate dehydrogenase (MDH) and peroxidase (PDX) are possible to be used as stress markers in in vitro conditions. Our results confirm earlier findings about negative effects of hyperhydricity on in vitro plant morphogenesis and ultrastructure, which in eucalypt is associated with a stressful condition contributing to lower propagation ratios.
The influence of increasing concentrations of naphthaleneacetic acid and the antibiotics cefotaxime, timentin, kanamycin, and hygromycin on eggplant (Solantun melongena L. cv. Embil) somatic embryogenesis was investigated. Cotyledon explants were excised from 16 to 20 days old in vitro grown seedlings. NAA promoted somatic emb...ryogenesis, although its concentrations had no influence on the mean number of embryos. Callusing decreaSed significantly with increasing NAA concentrations. Morphogenesis was stopped with 50 to 100 mg L-1 kanamycin and 7.5 to 15 mg L-1 hygromycin. Although early globular embryos were observed up to 15 mg L-1, further embryo development was inhibited at 10 mg L-1. Interestingly, cefotaxime (250 and 500 mg L-1) promoted a marked effect on enhancing fresh weight of calli, accompanied by decrease in embryo regeneration, whereas timentin concentrations (150 and 300 mg L-1) did not affect embryo differentiation as compared to the control treatment.
The world industry of ornamental plants is under wide transformations regarding the changes in the cultivation, management, postharvest technologies and the use of new (bio)technologies to assist the improvement and development of new varieties. This industry worth more than US$ 20 billion, but Brazil shares only 1% of this value. To compete...at this market with permanent technological innovation, the country needs to be tuned in with the progresses in the biotechnologies applied to the productive chain of ornamental plants. In this revision, we analyze the Brazilian and world ornamental market emphasizing the role of the biotechnologies in the modernization and increase of the competitiveness of the sector.
Cotyledon protoplasts were isolated from 16-18-day-old in vitro grown seedlings of 9 Brazilian and 3 Hungarian pepper varieties and hybrids. Large numbers (average 9.59 X 106 protoplasts g 14 fresh weight) of highly viable (average 87.0%) protoplasts were released using a pectocellulolytic enzyme mixture. Protoplasts were cultured in... K8p mediuni using an alginate disc embedding method. The osmotic pressure of the medium surrounding the alginate-embedded protoplasts was reduced by replenishing the liquid medium at K8p:K8 ratios of 1:0. 2:1, 1:1 in the first. second, and third week, respectively. Initial plating efficiency (IPE) average was 38.5% and after 21 days protoplasts reached microcolonies (15-20 cells) stages. Microcolonies were transferred after 3-4 weeks to a MS-based medium supplemented with 1.0 mg I-1 zeatin, 3.0% (w/v) sucrose, 0.24% (w/v) phytagel and pH 5.8, whereupon they formed callus. Final plating efficiency (FPE) average was 0.29% at a plating density of 1.0 x 105 protoplasts Protoplast-derived calli were cultured on a range of MS-based media supplemented with either BAP, IAA, TDZ; and zeatin. No morphogenic response was observed in any genotype investigated.