Somatic embryogenesis from stigmas and styles of grapevine

Summary An in vitro protocol has been developed for callus indiction, somatic embryogenesis, and plant regeneration from stigma-style culture of grapevine. Four different grapevine cultivars (Vitis vinifera L.: cvs. ‘Bombino Nero’, ‘Greco di Tufo’, ‘Merlot’, and ‘Sangiovese’) were tested. Exlants were cultured on Nitsch and Nitsch medium (NN) supplemented with various combinations of 6-benzylaminopurine (BA: 4.5 and 9.0 μM) and β-naphthoxyacetic acid (NOA; 5.0 and 9.9 μM). Sucrose (88 mM) was used as the carbon source. Somatic embryogenesis was induced within 3–7 mo. after culture initiation. Even though explants of different origin (unfertilized ovules and anthers) regenerated somatic embryos, the higher embryogenic potential was observed in stigma and style explants, with the exception of ‘Merlot’, which regenerated somatic embryos only from unfertilized ovules. The percentages of stigma-style explants producing somatic embryos was 7% in ‘Bombino Nero’ (cultured on NN medium supplemented 9.0 μM BA and 9.9 μM NOA). 14% in ‘Greco di Tufo’ (4.5 μM BA and 9.9 μM NOA), and 8% in ‘Sangiovese’ (9.0 μM BA and 9.9 μM NOA). The presence of growth regulators (BA and NOA) in the medium was essential for induction of somatic embryogenesis. Plants were regenerated on hormone-free NN medium containing 88 mM sucrose.     

Somatic embryogenesis and plant development from anther culture of Vitis vinifera (L.)

Anthers from Frumoasa alba (White beauty), Otilia, Valerien, Mission and Siegfried Rebe (FS₄) cultivars were cultured at the uninucleate stage of the microspore on Murashige and Skoog (1962) and Nitsch and Nitsch (1969) media supplemented with 2,4-dichlorophenoxyacetic acid (4.9 M) and benzyladenine (4.4 M). The primary calli were subcultured on MS medium with 6.6 M BA and 1.1 M indolylacetic acid, in order to induce their growth and plant regeneration. After seven months, vegetative buds were obtained with Frumoasa alba (2.7%), Otilia (0.3%), Valerien (4.5%), embryogenic callus was obtained with Mission and plant regeneration with Siegfried Rebe. Long term embryogenesis was maintained in Mission cv. for four years, by selection and regular transfer of the embryogenic areas of anther-derived calli. The embryogenic calli have the ability to generate abnormal somatic embryos with one, two or three cotyledons and cup or trumpet-shaped with fused cotyledons. In parallel with the embryogenic process, organogenesis with buds, leaf and shoot differentiation was regularly observed.     

Aneuploid plants derived from crosses with triploid grapes through immature seed culture and subsequent embryo culture

Through immature seed culture and subsequent embryo culture, aneuploid plants were derived from various crosses among 184 different triploid hybrid grape vines. In self-pollinations of the 184 vines, 0 to 1.6% of flowers produced immature seeds. In 16 reciprocal crosses between diploid and triploid and between tetraploid and triploid grapes, 0 to 23.0% of flowers produced immature seeds. The immature seeds excised 30–50 days after pollination were cultured for three months on Nitsch and Nitsch medium supplemented with L-glutamine, L-serine, L-cysteine and casein hydrolysate. Embryos developed within the cultured immature seeds were subcultured onto germination medium consisting of MS medium with 1 μM BA. Thirty-four of 137 embryos from 458 immature seeds germinated. Five of the 34 embryos grew normally. The five recovered plants were aneuploids with chromosome numbers from 51 to 59. The rates of embryo and plant recovery were different in different crosses with triploid grapes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Progress in grapevine breeding

Summary The European, or bunch grape, Vitis vinifera, is widely grown because of its high fruit quality and its capacity to grow in a wide range of climatic conditions. However, they are susceptible to fungal diseases and insect pests, especially when grown in cool, wet climates. The aim of a number of grapevine breeding programs throughout the world is to develop new varieties resistant to diseases using complex hybrids between European and American species of Vitis. Within these breeding programs it is essential to maintain heterozygosity and desirable hybrids are multiplied by asexual propagation. New approaches to grapevine improvement include the use of protoplast fusion to overcome sexual barriers, however the routine regeneration of plantlets from protoplasts and calluses is difficult. In vitro rescue of ovules from varieties with stenospermocarpic seeds shows considerable promise for breeding new seedless grapes. Eventually the use of plant transformation techniques to insert specific pieces of DNA coding for desirable genetic characteristics will provide opportunities for equipping well known grape cultivars with new characteristics.     

Direct seeding of grapevine somatic embryos and regeneration of plants

Summary Somatic embryos of grapevine (Vitis vinifera L.) ‘Chardonnay’ were produced from liquid suspension cultures. Mature somatic embryos were blot dried briefly in the laminar flow hood and germinated directly in Magenta GA-7 Vessels^™ containing one of the following potting media: (1) sand, (2) commercial potting mixture (CPM), or (3) CPM overlaid with sand. Each vessel containing 20 ml of distilled water and the potting medium was sterilized by autoclaving for 30 min and cooled overnight before inoculating the somatic embryos. Five somatic embryos were placed in each vessel under aseptic conditions. The vessels were closed and incubated at 26±2°C, 16 h photoperiod at 75 μmol s⁻¹ m⁻² light intensity. Results revealed that CPM overlaid with sand was best for plant development. There was more contamination of somatic embryos on pure CPM. Since direct seeding bypasses at least two subcultures in agar medium, it has implications for use of somatic embryos as ‘synthetic seeds’ for clonal plant production. This study shows that somatic embryos of grapevine can be handled with some of the convenience of seeds, emphasizing the feasibility for further automating in vitro plant production, which might be especially useful for new varieties where propagation material is limited.     

Anthocyanic vacuolar inclusions (AVIs) selectively bind acylated anthocyanins in Vitis vinifera L. (grapevine) suspension culture

Anthocyanic vacuolar inclusions (AVIs) appear as dark red-to-purple spheres of various sizes in vacuoles of grapevine (Vitis vinifera L.) cell suspension culture due to their interaction with anthocyanins. AVIs were purified and the bound anthocyanins extracted and analysed by HPLC from two lines of V. vinifera isolated from the same callus accumulating anthocyanin in the dark, yet varying in their anthocyanin profiles and accumulation. An intermediate-pigmented line (FU-1) with a 1.3:1 ratio of acylated:non-acylated anthocyanins, a colour value of 0.84 units and cyanidin and peonidin as the dominant species was compared with a high-pigmented line (FU-2) with a 1.2:1 ratio of acylated:non-acylated anthocyanins, a colour value of 3.72 units and malvidin predominating. The profile of AVI-bound anthocyanins showed an increase in acylated anthocyanins in both lines of approx. 28–29%, with no apparent preference for anthocyanin species. This resulted in a ratio of acylated:non-acylated anthocyanins of 6.2:1 for FU-1 and 4.9:1 for FU-2. The reasons for the selectivity of the AVIs for acylated (specifically p-coumaroylated) species compared with the whole cell profile are discussed.     

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B₁ peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca²⁺-channel blockers Co²⁺, Cd²⁺ and La³⁺, and was counteracted by Ca²⁺ ions, but was not reversed when the Ca²⁺-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg²⁺ ions but not by Sr²⁺, and was accentuated by Ba²⁺ ions. These results suggested that Ca²⁺ and Mg²⁺ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca²⁺/Mg²⁺-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.     

Polyamines in grapevine microcuttings cultivated in vitro. Effects of amines and inhibitors of polyamine biosynthesis on polyamine levels and microcutting growth and development

The main free amines identified during growth and development of grapevine microcuttings of rootstock 41 B, (Vitis vinifera cv. Chasselas × Vitis berlandieri) cultivated in vitro were agmatine, putrescine, spermidine, spermine, diaminopropane and tyramine (an aromatic amine). Amine composition differed according to tissue, with diaminopropane the major polyamine in the apical parts, internodes and leaves. Putrescine predominated in the roots. There was also a decreasing general polyamine and specific tyramine gradient along the stem from the top to the bottom. Conjugated amines were only found in roots. The application of exogenous amines (agmatine, putrescine, spermidine, tyramine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these amines can be growth limiting. Diaminopropane (the product of oxidation of spermidine or spermine by polyamine oxydases) strongly inhibited microcutting growth and development. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), led to inhibition of microcutting development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition indicating that polyamines are involved in regulating the growth and development of grapevine microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis from ornithine decarboxylase (ODC), had no effect on microcutting development and growth. We propose that ADC regulates putrescine biosynthesis during microcutting development.     

Long-term study of somatic embryogenesis from anthers and ovaries of 12 grapevine (<Emphasis Type="Italic">Vitis</Emphasis> sp.) genotypes

Summary Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.     

Low temperature storage of suspension culture-derived grapevine somatic embryos and regeneration of plants

Summary A method of clonal germplams preservation utilizing dehydrated somatic embryos and cool temperature storage conditions was demonstrated. Somatic embryos of grapevine (Vitis vinifera L) Autumn Seedless and Chardonnay were produced from suspension cultures. After washing twice with sterile water mature somatic embryos were blot-dried and placed on sterile filter paper in an open Petri dish in a laminar flow hood until they reached about 25% of their initial weight. Approximately 300 dried embryos were placed in each sterile 90×15 mm Petri dish, which was tightly sealed with two layers of Parafilm^TM. Sealed dishes were stored in the dark at 4°C in a standard refrigerator. Samples of 25–60 individual dehydrated somatic embryos were periodically tested for viability by placing them on solidified MS medium for germination and plant regeneration. After 42 mo. of dehydrated storage, 90% of the somatic embryos regenerated into plants. To further test utility, of this storage method dehydrated embryos stored for 12 and 26 mo. were shipped from Florida to Washington where 75 and 87.5% regenerated into plants, respectively. Cool temperature storage of dehydrated somatic embryos is a simple and inexpensive method of clonal, germplasm preservation when compared to alternatives such as cryopreservation.     

Somatic embryogenesis in cotton (Gossypium). II. Requirements for embryo development and plant regeneration

Calli of cotton (Gossypium hirsutum L.) initiated from seedling hypocotyl tissue were placed in liquid suspension and maintained by serial subculture in hormone-free Murashige and Skoog (MS) medium. Suspensions were sieved and globular embryos collected, washed, resuspended in basal medium and plated onto various semi-solid media. High inorganic salts (MS), low salt (2/3 MS), excess KNO₃, and the growth regulators napthaleneacetic acid (NAA), gibberellic acid (GA₃) and kinetin were tested for their effects on somatic embryo maturation. Long-term embryo proliferation and maturation were best on medium containing MS plus 1.9g/l KNO₃. Embryos 3 mm to 10 mm in size were removed from this plating medium and placed on sterile vermiculite saturated with Stewart and Hsu's medium plus 0.1 mg/l indoleacetic acid (IAA). Plants were recovered from 10.6% of the embryos. When 5 mm embryos were placed on this medium, 30% of the embryos formed plants within six weeks. Smaller embryos required a longer period of development on the vermiculite and the addition of fresh medium supplemented with 0.1 mg/l GA₃. Plants with an extensive root system and two true leaves were removed from sterile culture and potted in either one-to-one peat and sand, or vermiculite. Eighty percent of the regenerants were successfully hardened when glass beakers of increasing size (10 to 150 ml) were sequentially placed over the young plants during a two-week period.     

Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.     

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm⁻³ α-naphthalene acetic acid (NAA) + 0.5 mg dm⁻³ 6-benzyladenine (BA) for embryos production and 0.03 mg dm⁻³ NAA + 0.5 mg dm⁻³ BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.     

Antagonistic actinomycetes from Moroccan soil to control the grapevine gray mold

One hundred and forty-two different actinomycete strains were isolated from rhizosphere soil of Vitis vinifera L. sampled from four Moroccan areas. To evaluate the antifungal effect of the different collected actinomycete isolates, five fungi known to be phytopathogens (Pythium ultimum, Fusarium oxyysporum f. sp. albedinis, Sclerotium rolfsii, Verticillium dahliae and Botrytis cinerea) were used. Results showed that 24 isolates had an in vitro inhibitory effect toward at least 4 of the indicator fungi, but only 9 inhibited all these phytopathogens. These nine isolates were subsequently evaluated individually using in vitro grapevine plantlets for their ability to protect against plant gray mold. We demonstrate here that pre-inoculation of plantlets with these isolates allow them to withstand Botrytis cinerea. Six of these strains were shown to belong to the genus Streptomyces and three to the genus Micromonospora. These findings indicate the potential of developing effective actinomycetes from Moroccan habitats for the biological control of Botrytis cinerea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Somatic embryogenesis in Cucurbitaceae

Strategies based on the application of biotechnologies to crop improvement programmes generally require regeneration of whole plants from cells or tissues cultivated in vitro. In Cucurbitaceae, regeneration can occur either through a caulogenic or an embryogenic developmental pathway. Reports of somatic embryogenesis have dealt with the main cultivated crops, i.e. cucumber, melon, squash and watermelon. Somatic embryogenesis and plant recovery are obtained from numerous sources including protoplasts, but the best results are observed with explants coming from seedlings, especially cotyledons and hypocotyls. The genetic constitution of mother plants also seems to play a key role in the success of embryogenesis, but few systematic studies on genotype effect have been published. Somatic embryos can exhibit developmental abnormalities, particularly when they arise from protoplast-derived cultures. Generally, data concerning embryo yield, rate of germination and plant development and characteristics of regenerated plants and their progeny, has not been provided in previous reports. The potential use of somatic embryogenesis in cucurbit breeding programmes is stressed in this review.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - B₅ Gamborg et al. (1968) - CH casein hydrolysate - CW coconut water - 2,4-d 2,4-dichlorophenoxy acetic acid - GA₃ gibberellin A₃ - GA₄ gibberellin A₄ - H Heller (1953) - IAA indole 3 acetic acid - IBA indole 3 butyric acid - 2ip 2 isopentenyladenine - KIN kinetin - MS Murashige & Skoog (1962) - N Nitsch (1951) - N6 Chu et al. (1975) - NAA 1 naphthalene acetic acid - TDZ thidiazuron - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Levels of 4-hydroxystilbene-oxidizing isoperoxidases related to constitutive disease resistance in in vitro-cultured grapevine

A zymographic screening of peroxidases (EC 1.11.1.7) capable of oxidizing 4-hydroxystilbene was carried out by means of the peroxidase-catalyzed oxidative coupling of 4-hydroxystilbene and 4-aminoantipyrine. This screening reveals that only a few isoperoxidases are active in oxidizing 4-hydroxystilbene to viniferin-type compounds in in vitro cultures of grapevine. Unlike total peroxidase activity measured with 4-methoxy--naphthol, the levels of total peroxidase activity measured using 4-hydroxystilbene are related to disease resistance against downy mildew in axillary bud cultures of Vitis vinifera and (Vitis vinifera x Vitis rupestris) x Vitis riparia. From this observation, and using the above zymographic assay, it was found that a 4-hydroxystilbene-oxidizing isoperoxidase was overexpressed in both leaves and stems of the hybrid in relation to the increase in disease resistance of this species. These results suggest that constitutive 4-hydroxystilbene-oxidizing isoperoxidases may participate through their role in viniferin synthesis in the constitutive resistance mechanism that grapevines show against downy mildew.Abbreviations 4-AAP 4-aminoantipyrine - HRP horseradish peroxidase - 4-HS hydroxystilbene - HSPrx 4-hydroxystilbene-oxidizing peroxidase - 4-MN 4-methoxy--naphthol     

Somatic embryogenesis in Nicotiana tabacum L.: induction by thidiazuron of direct embryo differentiation from cultured leaf discs

Summary Induction of somatic embryogenesis by different growth regulators was examined in leaf disc cultures of Nicotiana tabacum L. Direct differentiation of somatic embryos occurred on media supplemented with naphthaleneacetic acid (NAA) and N⁶-benzylaminopurine (BAP). Thidiazuron (N-phenyl-N-1, 2,3,-thiadiazol-5-ylurea; TDZ) not only substituted for the most effective NAA-BAP combination but also induced a higher frequency of somatic embryogenesis. Regenerated somatic embryos were capable of developing into plants.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - NAA Naphthaleneacetic acid - TDZ N-phenyl-N-1,2,3,- thiadiazol-5-ylurea     

Study of genetic relationships between wild and domesticated grapevine distributed from Middle East Regions to European countries

Archaeobotanical-archaeological, cultural and historical data indicate that grapevine domestication can be dated back from 6000 to 7000 years ago and that it took place in the Caucasian and Middle East Regions. However, events leading to the domestication of this crop species are still an open issue. In this paper, 6 chloroplast microsatellites have been used to assess genetic similarities among, and within, domesticated and wild grapevine accessions representative of 7 distinct geographical regions from the Middle-East to Western Europe. Results show that 2 out of the 6 analyzed chloroplast loci are polymorphic within the 193 domesticated individuals and the 387 samples of 69 wild populations. Allele variants of the Cp-SSR loci combine in a total of 6 different haplotypes. The data show that the haplotype distribution is not homogeneous: the 6 haplotypes are present in the domesticated varieties, but only 5 (haplotype VI is absent) are observed in wild populations. The analysis of haplotype distribution allows discussion of the relationships between the two grape subspecies. The contribution of the wild grape germplasm to the domesticated gene pool still growing in different geographical regions can be, in cases, made evident, suggesting that beside domestication, gene introgression has also played a role in shaping the current varietal landscape of the European viticulture.

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