红将军苹果离体叶片高频再生体系研究

以中熟红富士品种红将军组培苗的叶片为试材,研究了离体叶片诱导再生不定芽过程中不同激素种类与组合、暗处理时间、取叶部位等因素对再生率的影响。结果表明,试管苗继代培养3～4代,当代培养25～30d,取顶部嫩叶3～4枚,剪除叶片边缘,将剪切的叶片中部以近轴面接触在最佳诱导分化培养基上:MS+TDZ1.0mg/L+IAA0.5mg/L+蔗糖30g/L,培养基pH值5.8,黑暗培养10d后,转入光照培养条件下,再生频率达100%,再生芽数平均4.6个。叶片再生不定芽分化后15～20d转接到苹果继代培养基MS+BA0.5mg/L+IBA0.1mg/L上,当不定芽长度约2cm时再转接到1/2MS+IBA0.5mg/L+NAA0.5mg/L+Ac0.5g/L+蔗糖2%的生根培养基上,光培养15d后,生根率可达86.0%。     

Optimisation of a highly efficient shoot regeneration system using leaf explants of Chinese jujube (Ziziphus jujuba Mill.) by response surface methodology

Chinese jujube (Ziziphus jujuba Mill.) is a major fruit crop in Asia. In this study, response surface methodology (RSM) was successfully employed to establish a highly efficient in vitro propagation and regeneration system for the ‘Teapot’ jujube via shoot organogenesis. Among the tested factors, gibberellic acid (GA₃) concentration showed the most significant positive effect. The pre-culture darkness timing and medium were also important factors for highly efficient shoot regeneration of the ‘Teapot’ jujube. The highest regeneration (> 75%) was achieved by 1 week in darkness and culture on wood plant medium (WPM) containing 0.25 mg·L^?1 GA₃, 0.5 mg·L^?1 6-benzylaminopurine (BAP) and 0.1 mg·L^?1 3-indoleacetic acid (IAA). In vitro-derived shoots rooted very well in the modified ¹/₂ Murashige and Skoog (MS) medium containing 0.4 mg·L^?1 3-indolebutyric acid (IBA), resulting in a 100% rooting rate. These findings suggest that the RSM can be employed to optimise the protocols needed for successful in vitro plant regeneration of jujube cultivars, with potential applications in plant genetic transformation practices, polyploidy induction and germplasm preservation.     

Effects of CO2 on greenhouse grown eggplant (Solanum melongena L.) II. Leaf tip chlorosis and fruit production

The effect of CO, on leaf boron content, Leaf Tip Chlorosis (LTC) and fruit production of eggplant (Solanum melongena L., cv. Cosmos) was investigated in the spring of 1991. Two levels of CO₂ (413 and 663 (xmol mol^-1) were maintained in duplicate, in four glasshouse compartments (16 m x 16 m). LTC was significantly more severe at high than at low CO₂. Leaf boron content was lower in leaves with LTC than in other leaves and was lower in leaves from high CO₂ than in those from low CO₂. These results, in combination with observed reduction in leaf conductance (part I), support the hypothesis that LTC is caused by reduced boron translocation to young, fast growing leaves, because of reduced transpiration. More specific research on boron is necessary to confirm this hypothesis. Fruit production was significantly higher (24%) at high CO₂ than low CO₂, despite more severe LTC.     

A comparison of the gelling of isubgol,agar and gelrite on in vitro shoot regeneration and rooting of variety Samsun of tobacco (Nicotiana tabacum L.)

Agar is being used to solidify media for plant tissue culture since long. Both purity and type of agar or gelling agent influence the behaviour and growth of tissues in culture. The study using leaf disc explants of variety Samsun of tobacco compares adventitous shoot regeneration on 0.5 cm × 0.5 cm leaf discs cultured on MSD4X2 medium and rooting on MSO medium gelled with different blends of agar–isubgol, gelrite–isubgol, phytagel–isubgol or isubgol singly. It was found that irrespective of some problems associated with isubgol gel, the maximum number of shoot per explant were recorded on MSD4X2 medium gelled with 7 g/l isubgol. The longest shoots were recorded on MSD4X2 medium gelled with 9 g/l isubgol. Likewise, the highest number of roots were also recorded on MSO medium gelled with 7 g/l isubgol. For a given quantity of a medium, agar, gelrite/isubgol blends are very cheap compared to agar used singly. Moreover, blends of agar/isubgol, gelrite/isubgol gelled at low temperatures indicated safe use of these gels for heat labile substances in genetic transformation or tissue culture experiments. The results emphasized the potential of the isubgol used singly or in combination with agar and gelrite for economic commercial application, replacing the costliest, though not indispensable, gelling agent agar.     

Effects of biennial cropping and controlled shoot density on yield performance and fruit quality in red raspberry (Rubus idaeus L.)

Effects of annual versus biennial cropping with varying shoot densities on plant structure, berry yield and quality were studied in ‘Glen Ample’ raspberry over a period of four seasons (two cropping years). In the vegetative phase, primocane height and internode length were larger in the annual than in the biennial cropping system. These parameters as well as Botrytis infestation increased with increasing shoot density. In both cropping years, berry yields per unit area were about 20% higher in the biennial cropping system, whereas yields per shoot were not significantly different in the two systems. In both cropping systems, yields per shoot strongly declined with increasing shoot density, while yields per metre row increased slightly. Regardless of cropping system, yields per metre row did not increase with increasing shoot density beyond eight shoots per metre. The concentrations of dry matter, soluble solids, titratable acidity and ascorbic acid as well as the intensity of juice colour all declined with increasing shoot density. We conclude that under controlled shoot density conditions, there is little scope for biennial yield increases that fully compensates for the lost crops every second year. However, the system greatly facilitates berry harvest and eases plant disease pressure.     

Development of a regeneration protocol through indirect organogenesis in prickly pear cactus (Opuntia ficus-indica (L.) Mill)

An indirect organogenesis regeneration protocol for Opuntia ficus-indica (L.) Mill var “Blanco sin Espinas” is described. One centimeter square cladode explants sections from previously micropropagated prickly pear plants were cultured in Murashige and Skoog (MS) basal medium supplemented with 20 different combinations of 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyladenine (BA). The best calli induction and regeneration response were observed when 2.26 μM 2,4-D and 2.21 μM BA combination was applied to the nopal explants. Regenerating calli was capable of forming new buds when transferred to MS basal medium supplemented with 0.5 μM BA (proliferation medium). Shoot elongation and rooting were achieved on MS medium without plant growth regulators. Excellent acclimatization to greenhouse conditions was observed for all transferred plantlets. By this procedure no morphological differences were observed between the regenerated and mother plants. This protocol may be also utilized to carry out plant regeneration after genetic transformation, in order to develop transformed plants without the presence of chimeric zones.     

Adventitious shoot regeneration from hypocotyl slices of mature apricot (Prunus armeniaca L.) seeds: A feasible alternative for apricot genetic engineering

Adventitious shoot regeneration from hypocotyl slices of mature apricot seeds has been achieved with regeneration percentages of 31.7%, 44.4%, and 46.9% for the cultivars ‘Canino’, ‘Dorada’, and ‘Moniqui’, respectively. Regeneration was significantly affected by the parental origin of the explants (P < 0.05) but not by thidiazuron or 3-indolebutyric acid for any of the three cultivars, at the levels tested. None of the other factors studied (basal medium, 2,4 dichlorophenoxy-acetic acid pulses, dark incubation period, or addition of silver thiosulfate) affected significantly shoot regeneration percentage from ‘Canino’ hypocotyl sections. The effect of paromomycin on regeneration was genotype-dependent and different dose–response curves were obtained for each cultivar. While 40 μM paromomycin completely inhibited regeneration from ‘Canino’ sections, some buds were obtained from ‘Dorada’ and ‘Moniqui’ explants. The two aminoglycoside antibiotics tested, kanamycin and paromomycin, showed differing toxicity on ‘Canino’. A lower concentration of kanamycin (20 μM) than of paromomycin inhibited totally adventitious regeneration from ‘Canino’ explants. Agrobacterium-mediated transformation experiments, with the non-oncogenic strain AGL1 harboring the binary plasmid p35SGUSINT, were performed and GUS assays were carried out after four weeks to determinate stable transformation events. The utilization of paromomycin (10 μM) as the selective agent increased significantly both the number of explants that presented at least one transformation event (P < 0.05) and the number of large area or calli expressing the gus gene (P < 0.001), compared with the addition of kanamycin (10 μM). Moreover, when 10 μM paromomycin was added to the medium some massively transformed explants were observed and a chimerical bud was regenerated.     

Effect of water deficit and rewatering on leaf gas exchange and transpiration decline of excised leaves of four grapevine (Vitis vinifera L.) cultivars

Grapevine cultivars are known to differ in their drought adaptation mechanisms, but there is little knowledge on how they behave when recovering after a drought event. The effects of increasing water deficit and recovery after rewatering were evaluated on four widely grown red grapevine cultivars native from different climates (Cabernet Sauvignon, Cs; Garnacha, syn. Grenache, Ga; Merlot, Me; and Tempranillo, Te) through the study of gas exchange (GE) measurements and transpiration decline curves (TDC). As a whole, Ga has proved to be the cultivar best adapted to water deficit, since it showed the highest water use efficiency (WUE) and the greatest water saving ability after leaf excision. Te, on the contrary showed the lowest values for those parameters under increasing stress, although when rewatered showed greater acclimation ability than Cs and Me, remarkably improving its behaviour. The two methodological approaches (GE and TDC) used at different water deficit levels and after rewatering, have complemented each other, allowing a better cultivar characterization than each method would had allowed itself.     

Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2

Although proline is one of the major computable organic solutes that accumulate in many plant species in abiotic stresses, a hot debate continues about whether proline accumulation is a reaction to abiotic stress, or a plant's response is associated with stress tolerance. The effects of proline on antioxidative system in grape leaves of Vitis vinifera L. cv., ‘Öküzgözü’ exposed to oxidative stress by H₂O₂ was investigated. Endogenous proline, hydrogen peroxide (H₂O₂), malondialdehyde (MDA) concentrations, percentage of electrolyte leakage (EL), and some of the antioxidant enzyme activities; such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) were measured spectrophotometrically. Inhibitory effect of H₂O₂ on antioxidant enzyme activities, MDA, and EL was found. In the presence of proline, SOD and CAT activities decreased, while POD and APX activities increased. Proline pre-treatment resulted in a decrease in cellular H₂O₂ content, MDA, and EL, while cellular concentration of proline increased. Based on the finding, it was suggested that proline and H₂O₂ could play an important role in oxidative stress injury of grapevine leaves grown in vitro culture. Also, proline might have a direct positive effect on antioxidant enzyme system, membrane phase change, MDA, and EL.     

Effects of exogenous application of plant growth regulators on the development of ovule and subsequent embryo rescue of stenospermic grape (Vitis vinifera L.)

Seedless grapevine cultivars (Vitis vinifera L.) are widely grown in Europe, America and Asia. Abortion of zygotic embryos in seedless grapes largely limits the efficiency of breeding of seedless cultivars through genetic crossing. The present study was designed to investigate effects of exogenously applied plant growth regulators (PGRs) to the grapevines in field condition on ovule and subsequent embryo rescue of seedless grapes of small seed traces. First experiment was performed by measuring ovules weight, proportion of each category ovules in maturity and embryo development in vitro of seedless grape cv. Centennial Seedless, Thompson Seedless and Crimson Seedless sprayed by chlormequat (CCC), benzyladenine (BA), ethephon (CEPA) and putrescine (Put). The effects of different application concentration and date of CCC were further evaluated in Centennial Seedless in later experiment. The results showed that exogenous application of all PGRs did not affect the total number of ovules per berries in maturity. CCC increased the ovules weight and proportion of ovules >4 mm in length of three varieties in maturity. The effects of two application times of PGRs on weight of berries and ovules and proportion of each category ovules in maturity were not significantly different. In the proceeding of embryo rescue, CCC at 100 and 1000 mg l⁻¹, BA at 100 mg l⁻¹ and Put at 20 mg l⁻¹ increased the percentage of developed embryos of Centennial Seedless and Thompson Seedless. The results showed that the size of ovules excised for embryo rescue significantly affected embryo formation and plant regeneration. The percentage of embryos formation in ovules >4 mm in length was significantly more than in ovules 2–4 mm in length, no embryo was found in ovules <2 mm in length. Exogenous application of CCC at 100–500 mg l⁻¹ significantly increased percentage of ovules >2 mm in length by 80.0–82.7% in Centennial Seedless, therefore improving embryo formation. The statistical correlation was found between the proportion of ovules >2 mm and embryo formation (r = 0.92) in Centennial Seedless. Among the different spraying time in Centennial Seedless, CCC applied 14 days before bloom produced significantly more ovules >2 mm in length and embryos formation.