Somatic embryogenesis in leaves and leaf-derived protoplasts of Actinidia deliciosa var. deliciosa cv. Hayward (kiwifruit)

Summary The obtention of embryogenic competence in Actinidia deliciosa var. deliciosa cv. Hayward is reported. Axillary buds from shoots submitted to cold (4°C) and starvation for 1.5 months, developed leaves with embryogenic competence. These leaves, cultured in darkness for 1.5 months on a medium containing zeatin as a sole growth regulator, originated compact structures from which embryos developed. The plating orientation and sectioning of leaves strongly affected the expression of the embryogenic potential. A selected fraction of the protoplasts isolated from these leaves was able to develop in an embryogenic way. The germination of the embryos is still only occasional.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 6-dimethylallyl aminopurine - IAA indole-3-acetic acid - NOA naphthoxyacetic acid - SEM Scanning Electron Microscopy     

Transformation of carotenoid biosynthetic genes using a micro-cross section method in kiwifruit (Actinidia deliciosa cv. Hayward)

Genetic transformation using a micro-cross section (MCS) technique was conducted to improve the carotenoid content in kiwifruit (Actinidia deliciosa cv. Hayward). The introduced carotenoid biosynthetic genes include geranylgeranyl diphosphate synthase (GGPS), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), β-carotene hydroxylase (CHX), and phytoene synthase (PSY). The transformed explants were selected on half-strength MS medium containing 0.001 mg l⁻¹ of 2,4-D and 0.1 mg l⁻¹ of zeatin, either 5 mg l⁻¹ hygromycin or 25 mg l⁻¹ kanamycin, and 500 mg l⁻¹ cefotaxime. The genomic PCR, genomic Southern blot analysis, and RT-PCR were performed to confirm the integration and expression of the transgenes. The transformation efficiencies of either kanamycin- or hygromycin-resistant shoots ranged from 2.9 to 22.1% depending on the target genes, and from 2.9 to 24.2% depending on the reporter genes. The selection efficiencies ranged from 66.7 to 100% for the target genes and from 95.8 to 100% for the reporter genes. Changes of carotenoid content in the several PCR-positive plants were determined by UPLC analysis. As a result, transgenic plants expressing either GGPS or PSY increased about 1.2- to 1.3-fold in lutein or β-carotene content compared to non-transgenic plants. Our results suggest that the Agrobacterium-mediated transformation efficiency of kiwifruit can be greatly increased by this MCS method and that the carotenoid biosynthetic pathway can be modified in kiwifruit by genetic transformation. Our results further suggest that GGPS and PSY genes could be major target genes to increase carotenoid contents in kiwifruit.     

Studies on the Somaclonal Variation of Regenerated Plants from Protoplasts of Actinidia deliciosa

Protoplasts were isolated from leaf, petiole and stem segment-derived calli induced from one pistillate plant of Actinidia deliciosa line No. 26. Regenerated plantlets were obtained from protoplasts of leaf-derived and stem segment-derived calli, while only calli regenerated from protoplasts of petiole-derived calli. Seventy-six plants from protoplasts of leaf-derived calli and 21 plants from that of stem segment-derived calli survived after transplanting or grafting during 1987–1989. One staminate plant and two pistillate plants bloomed in May, 1991 and fruited soon afterwards. In all of those three plants regenerated from protoplasts of leafderived calli, sex differentiation occurred from somatic cells of Actinidia was verified. Somaclonal variation on leaf shape and plant morphology was obviously appeared. Chromosome number identified from 16 plants varied from 116 to 180.     

Plant regeneration from protoplasts of long-term callus cultures of Actinidia deliciosa var. deliciosa cv. Hayword (Kiwifruit)

Plant regeneration of Actinidia deliciosa var. deliciosa cv. Hayword was obtained from protoplasts isolated from petiole derived long-term callus cultures. Protoplasts were cultured in liquid medium over agarose gelled medium. Regenerated green callus, plated on solid medium, could develop shoots that rooted spontaneously in hormone-less medium. The plants obtained are growing fast in soil and present a normal phenotype.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiotreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kin kinetin - MES 2-(N-morpholino) ethanesulphonic acid - MS Murashige and Skoog (1962) medium - NAA naphthalene-1-acetic acid - SH Schenk and Hildebrandt (1972) medium This Research was supported by JNICT and INIC     

Plantlet Regeneration from Leaf Protoplasts in Seedling of Actinidia eriantha Benth.

Newly extended leaves of in vitro seedling of Actinidia eriantha Benth. were used for protoplast isolation. Protoplasts were cultured in liquid MS medium (devoid of NH4NO3) supplemented with 1.0 mg/L 2, 4-D and 0.4 mol/L glucose. The plating efficiency after 3 weeks of culture was about 19.4 %. Protoplasts-derived cells divided sustainably and developed into calli of 2 mm in size in the original protoplast-culture-medium without adding fresh medium so to decrease the osmotic pressure. These calli regenerated shoots when being transfered to MS medium with 0. 5 mg/L zeatin and 0. 1 mg/L IAA. Regenerated shoots were rooted by immersion in 20 ppm IBA solution before culturing on half-strength MS medium devoid of growth regulators.     

A genome-specific repeat sequence from kiwifruit (Actinidia deliciosa var. deliciosa)

Summary Six members of a family of moderately repetitive DNA sequences from kiwifruit (Actinidia deliciosa var. deliciosa) have been cloned and characterized. The repeat family is composed of elements that have a unit length of 463 bp, are highly methylated, occur in tandem arrays of at least 50 kb in length, and constitute about 0.5% of the kiwifruit genome. Individual elements diverge in nucleotide sequence by up to 5%, which suggests that the repeat sequence is evolving rapidly. Homologous sequences were found in A. deliciosa var. chlorocarpa. The repeat sequence was not found under low stringency hybridization conditions in the diploid A. chinensis, the species most closely related to the hexaploid kiwifruit, or in eight other Actinidia species. However, homologous repeats were detected in a tetraploid species, A. chrysantha. The results provide the first molecular evidence to suggest that kiwifruit may be an allopolyploid species.     

Phosphate Transport across the Plasma Membrane of Wheat Leaf Protoplasts: Characteristics and Inhibitor Specificities

The kinetics and inhibitor specificities of phosphate transport across the plasma membrane of wheat leaf mesophyll protoplasts have been examined. Studies were also carried out on the effects of light and pH on phosphate transport and the plasma membrane electropotential. At pH 5.8 (30°C), protoplasts accumulated phosphate at the rate of 3.9 ± 0.2 nanomoles per milligram protein per hour. Phosphate uptake rates and inhibitor specificities for the leaf cell plasma membrane phosphate transporter were qualitatively similar to those observed with root protoplasts. Neither picrylsulfonic acid, or p-chloromercuribenzene sulfonate affected phosphate uptake significantly at 0.1 millimolar. Of all compounds tested, carbonyl cyanide-p-trifluoromethoxy phenylhydrazone was the most effective inhibitor of phosphate uptake (60% at 0.1 millimolar). Tribenzylphosphate inhibited uptake by 34% while dibenzylphosphate had no effect. The plasma membrane electropotential was found to be −37 ± 3 millivolts. Initiation of photosynthesis lowered the membrane potential to −39 ± 3 millivolts. Inhibition of phosphate uptake by 34% with the substrate analog tribenzylphosphate resulted in a measured membrane potential of −33 ± 3 millivolts. These changes in potential were not significant at the 5% probability level. Phosphate uptake rates remained constant under photosynthetic and nonphotosynthetic conditions. The utility of tribenzylphosphate as an inhibitor in plant systems is demonstrated.     

Plant Regeneration from Protoplasts of Actinidia chinensis Planch.

Protoplasts isolated from cotyledon callus line of A14N7 of Actinidia Chinensis Planch. were cultured in the improved NN-69 medium. First division of regenerated cells occurred during 7–10 days of culture, and percentage of the cell division was about 10% at day 20. The best result of protoplast culture was achieved when protoplasts were cukured in liquid medium at a density of 5× 104/ml, About 4 months, procoplast-derived calli were transferred stepwisely onto differentiation media where they developed into green compact calli, from which the perfect plants were regenerated.     

Embryology and Embryo Rescue of an Interspecific Cross Between Actinidia deliciosa cv. Haywardand A. eriantha

The study deals with an investigation of embryo and endosperm development in seeds of interspecific hybridization betwwn Actinidia deliciosa cv. Hayward (6X) and A. eriantha (2X) and an attempt to hybrid embryo rescure. The average seed number per fruit of hybrid com suitable culture media for in vitro embryo growth and subsequent growth of the seedlings are tested for hybrid embryos from normal and abortive seeds. The highest percentage of seed germination and the best growth of early seedlings are obtained op. MS medium supplemented with 0.5 ppm of IAA and 0.5 ppm of GA3; MS supplemented with 2 ppm of 2ip, 0.5 ppm of IAA and 0.5 ppm of GA3; MS supplemented with of 2ip and 0.5 ppm of GA3. When these embryos, as seedlings, were transferred onto MS supplemented with 0.5 ppm of GA3 and then MS without growth regulators, they readily develop into seedlings with normal leaves and roots, A lot of adventitious buds produced from hypocotyl of some normal and abortive embryos on MS supplemented with 2 ppm of BAP and Ms+ 0.5 ppm IAA+0.5 ppm GA3 further grow into hybrid plantlets. Although various percentage of embryos developed from abortive seeds also germinate, but when inoculated onto media as mentioned in Table l, a number of malformed seedlings form subsequently. The remaining embryos on all the media, however, show limited growth, and eventually either form callus or die. The nature of hybrid seedling is confirmed by cytological test. Half of them showed chromosome number as 4X=116 approx., the rest may have 19, 27, 30, 46, 120, 131, ect. In conclusion, according to embryological and cytological observation on hybrid seeds and seedlings: (I) the cross between Actinidia deliciosa cv. Hayward and A. eriantha appears interspecific incompatibility and compatibility to some extent, (2) normal hybrid seedlings are produced by normal hybrid embryos from normal seeds and abnormal embryos from abortive seeds, and (3) both may induce initiation of adventitious buds from hypocotyl. We suggested that these results may be useful for plant breeding program.     

Plasma Membrane H+-ATPase in Guard-Cell Protoplasts from Vicia faba L.

The activity of plasma membrane H⁺-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO₃^–and vanadate. In the presence of oligomycin, ammonium molybdateand NO₃^–, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca²⁺, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theK_m was 0.5 mM for ATP. The activity required Mg²⁺ but was notaffected by K⁺, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)^–1.h^–1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H⁺-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987)     

Analysis of Glucocerebrosides of Rye (Secale cereale L. cv Puma) Leaf and Plasma Membrane

Glucocerebrosides of whole rye (Secale cerale L. cv Puma) leaf and plasma membrane were analyzed using gas chromatography-mass spectrometry and gas chromatography following hydrolysis or as intact molecules purified by reverse-phase high performance liquid chromatography. Fatty acids of acid-hydrolyzed leaf and plasma membrane glucocerebrosides consisted of >98 weight percent saturated and monounsaturated 2-hydroxy fatty acids which contained 16 to 26 carbon atoms. The major fatty acids detected were 2-hydroxynervonic acid (24:1h), 2-hydroxylignoceric acid (24:0h), 2-hydroxyerucic acid (22:1h), and 2-hydroxybehenic acid (22:0h). Long-chain bases of alkaline-hydrolyzed glucocerebrosides consisted primarily of cis-trans isomers of the trihydroxy base 4-hydroxysphingenine (t18:1) and the dihydroxy base sphingadienine (d18:2) with lesser amounts of 4-hydroxysphinganine (t18:0) and isomers of sphingenine (d18:1). Intact, underivatized glucocerebroside molecular species of rye leaf and plasma membrane were separated into more than 30 molecular species using reverse-phase HPLC. The molecular species composition of leaf and plasma membrane were quantitatively and qualitatively similar. The major molecular species was 24:1h-t18:1 which constituted nearly 40 weight percent of leaf and plasma membrane extracts. Several other species including 22:1h-t18:1, 24:1h-t18:1 (isomer), 22:0h-t18:1, 24:1h-d18:2, and 24:0h-t18:1 each comprised 4 to 8% of the total. It is anticipated that the high performance liquid chromatography procedure developed in this study to separate intact, underivatized lipid molecular species will be useful in future studies of the physical properties and biosynthesis of plant glucocerebrosides.     

Anatomy and Histochemistry of the Root System of the Kiwifruit Vine, Actinidia deliciosa var. deliciosa.

The kiwifruit vine is a species which has been newly introducedinto cultivation and little is known of its comparative physiologyand anatomy. In this study we found that fibrous, 'magnolioid'roots, which have undergone secondary vascular development butwhich retain the cortex and develop a suberized epidermis, comprisethe greater part of the root system (95% of total length). Newlyinitiated roots with primary development conform to norms establishedin other woody plant species. However, the structural roots,like the fibrous roots, also retain a cortex and phellodermwhich is initiated by hypodermal cells within the cortex andnot by the pericycle which is the common progenitor tissue inother species. This phellogen produces new cells centrifugallyonly. The cortex is a relatively small component of the structuralroot and the bulk of the tissue is vascular in origin, as inthe roots of other plant species. The endodermis is retainedand continues to divide periclinally to accommodate the increasein circumference with growth.Copyright 1993, 1999 Academic Press Actinidia deliciosa, root anatomy, ontogony, histochemistry, exodermis, endodermis     

ATP-Dependent Regulation of an Anion Channel at the Plasma Membrane of Protoplasts from Epidermal Cells of Arabidopsis Hypocotyls

Although Arabidopsis is the object of many genetic and molecular biology investigations, relatively few studies deal with regulation of its transmembrane ion exchanges. To clarify the role of ion transport in plant development, organ-and tissue-specific ion channels must be studied. We identified a voltage-dependent anion channel in epidermal cells of Arabidopsis hypocotyls, thus providing a new example of the occurrence of voltage-dependent anion channels in a specific plant cell type distinct from the stomatal guard cell. The Arabidopsis hypocotyl anion channel is able to function under two modes characterized by different voltage dependences and different kinetic behaviors. This switch between a fast and a slow mode is controlled by ATP. In the presence of intracellular ATP (fast mode), the channels are closed at resting potentials, and whole-cell currents activate upon depolarization. After activation, the anion current deactivates rapidly and more and more completely at potentials negative to the peak. In the absence of ATP, the current switches from this fast mode to a mode characterized by a slow and incomplete deactivation at resting potentials. In addition, the whole-cell currents can be correlated with the activity of single channels. In the outside-out configuration, the presence of ATP modulates the mean lifetimes of the open and closed states of the channel at hyperpolarized potentials, thus controlling its open probability. The fact that ATP-dependent voltage regulation was observed in both whole-cell and outside-out configurations suggests that a single type of anion channel can switch between two modes with distinct functional properties.     

Pollen Development in Actinidia deliciosa var. deliciosa: Histochemistry of the Microspore Mother Cell Walls

The development of the microspore mother cell walls in Actinidiadeliciosa (kiwifruit) has been studied using light and electronmicroscopy. The microspore mother cell wall is similar, histochemically,and structurally in anthers from both functionally staminateand functionally pistillate flowers. Deposition, which beginsduring early prophase I, produces an electron-dense multilaminatedwall layer (layer a) and by the end of meiosis I a thick electron-lucentlayer (layer b) to the inside of this multilayered wall. Thereasons for histochemical differences and similarities betweenthese layers are discussed. The original primary wall persistsuntil the late uninucleate microspore stage. Layer (b), whichis probably mainly callose, dissolves at the late tetrad/earlymicrospore stage while layer (a), which probably also containsother polysaccharides, persists and dissolves concurrently withthe primary wall. Actinidia deliciosa, kiwifruit, microspore mother cell wall, callose, histochemistry, light microscopy, electron microscopy, male sterility     

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.     

Plant Regeneration from Callus Protoplasts of Codonopsis pilosula

Embryogenic cell line was established from hypocotyl segments of Codonopsis pilosula (Franch.)Nannf. 4--8 day old embryogenic callus was used to isolate protoplasts in an enzyme solution containing 1.5 % cellulase Onozuka R-10 and 3 % pectinase. Protoplasts were cultured in MS,C81V,DPD and KMSp basal medium supplemented with 1.2 mg/L 2,4-D, 0.2 mg/L NAA, 0. 2 mg/L BAP, 0. 1 mg/L ZT,and different combinations of glucose and mannitol . Protoplast-derived cells underwent sustained divisions in KM8p medium. As an osmoticum, glucose was more beneficial to protoplast division. A combination of 0. 30 mol/L glucose with 0.10 mol/L mannitol gave the best result. Under proper conditions , protoplasts underwent the first division on the 3rd day of culture,formed colonies within 30 days , and developed into microcalli in 6 weeks. Plantlets were regenerated from protoplast-derived calli through somatic embryogenesis. 0.2 % activated charcoal promoted embryoid formation and root development.     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.