Somatic embryogenesis from grapevine cells. I-Improvement of embryo development by changes in culture conditions

In conventional culture conditions without auxin, somatic embryos arising from suspension cultures of grapevine rootstock 41B (Vitis vinifera cv. Chasselas x Vitis berlandieri) are arrested at the heart stage of development. Starting from indications that inhibitors excreted in the culture medium could be responsible for this arrest, new culture conditions based on daily subculturing embryos in fresh medium have been successfully used to obtain full embryo development. From this technique, a microassay was devised for screening small amounts of extracellular molecules as potential inhibitors of embryonic development. Our results show that extracellular macromolecules of molecular weight higher than 10 kDa are likely involved in the inhibition of caulinary meristem initiation. However, other factors obviously cooperate to inhibit embryo development in conventional culture conditionsAbbreviations CH76 cv. Chardonnay clone 76 (Vitis vinifera) - NOA 2-naphthoxyacetic acid     

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.     

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.     

Somatic embryogenesis from styles of lemon (Citrus limon)

Lemon [Citrus limon (L.) Burm.] styles were treated with different growth regulators for induction of somatic embryos. Styles and stigmas were dissected from flowers and cultured on a Murashige and Skoog (MS) basal medium supplemented with 4.52 M 2,4-dichlorophenoxyacetic acid and 13.3 M 6-benzyladenine. Callus was induced from the style base 2 weeks after the treatment initiation, and embryos appeared 2 months later.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid     

Somatic embryogenesis and plant regeneration from leaf tissue of jojoba

A protocol was developed for the induction, maturation and germination of somatic embryos from leaf tissue of jojoba [Simmondsia chinensis (Link) Schneider]. Explants were placed on their adaxial sides in Petri dishes and maintained in darkness on half-strength Murashige and Skoog basal medium (MS/2). Combinations of 2,4-dichlorophenoxyacetic acid (1.35–4.52 μM) with 6-benzylaminopurine (1.33–4.43μM) and 2 synthetic cytokinins, N-(2-chloro-4pyridyl)-N′-phenylurea (1.21–4.03μM) or (E)-6-[3-(trifluoromethyl)-but-2-enylamino] purine (1.11–3.71μM) resulted in formation of embryogenic cultures and somatic embryos. After two 30-day subcultures, embryogenic cultures were transferred onto MS/2 medium supplemented with different auxins and cytokinins. Somatic embryo maturation, germination and plantlet formation were achieved using 1-naphthaleneacetic acid (3.75μM) or indole-3-butyric acid (3.44μM) in combination with BA (0.44 or 1.33μM) or F3iP (0.37 or 1.11μM). Histology confirmed each stage of development. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Somatic embryogenesis and plant regeneration from petioles of Parthenocissus tricuspidata planch

Summary A protocol of somatic embryogenesis and plant regeneration from petiole segments of Parthenocissus tricuspidata Planch. has been developed. Embryogenic tissue was induced on B5 (Gamborg) basal medium supplemented with 2.25–9.0 μM 2,4-dichlorophenoxyacetic acid, 500 mg l⁻¹ casein hydrolysate (CH), and 0.1 gl⁻¹ activated charcoal. Somatic embryos were induced on B5 medium containing various concentrations of benzyladenine (BA) (4.44, 6.66, and 8.88 μM) and α-naphthaleneacetic acid (NAA) (0, 0.54, and 1.61 μM) plus 500 mg l⁻¹ CH. Ninety percent of normal somatic embryos were converted into plantlets directly on Murashige and Skoog (MS) medium free of plant growth regulators. Shoots could be induced from abnormal somatic embryos on MS medium containing 4.44 μM BA, 0.05 μM NAA, and 500 mg l⁻¹ CH. Genotypic differences were found in the process of somatic embryogenesis and plant regeneration. Histological analysis confirmed the process of somatic embryogenesis. Regenerated plantlets with well-developed roots were successfully acclimatized in greenhouse and all plants showed normal morphological characteristics.     

Somatic embryogenesis from callus cultures ofNardostachys jatamansi

Callus cultures ofNardostachys jatamansi DC, an endangered medicinal and aromatic plant, were established using petiole explants on MS medium supplemented with 16.1 µM -naphthaleneacetic acid and 1.16 µM kinetin. Embryogenesis in these callus cultures took place only upon sequential subculture of the callus on media having gradually decreasing auxin (16.1 to 1.34 µM NAA) and simultaneously increasing cytokinin (1.16 to 9.30 µM kinetin) concentrations over a period of 7 months. Somatic embryo to plantlet conversion took place on a medium containing 9.30 µM kinetin and 1.34 µM NAA.     

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.     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

Improved somatic embryogenesis of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) with focus on induction parameters and efficient plant regeneration

A study of four parameters (induction medium, floral explant, developmental stage and year) was carried out to determine the best combination for the embryogenesis induction of eight grapevine (Vitis vinifera L.) cultivars. Anthers and ovaries were extracted from flower buds at three developmental phases and incubated in two induction media over two consecutive years. As average, the percentage of embryogenesis on Nitsch and Nitsch-derived medium (9.1%) was higher than in Murashige and Skoog-derived medium (5.9%) and embryogenesis from ovaries (10.1%) was 2-fold higher than from anthers (4.9%). Earlier flower developmental stages (II–III) favored embryogenic induction from anthers, while later stages (III–V) did it from ovaries. Induction of embryogenic cultures was genotype dependent. Two years after the establishment of the embryogenic lines, an average of 48.0% of the pro-embryogenic masses were viable and suitable to initiate cell suspensions. Embryogenic cultures of four genotypes showed a high percentage of conversion from embryos to plants: Albariño (61.8%), Garnacha (48.8%), Tempanillo (71.0%) and Sultanina (69.0%). Moreover, cell suspensions were competent for transient transformation based on β-glucuronidase assay, as up to 6,387 blue spots per Petri plate after Biolistic bombardment were obtained. Here, we present the advantage of ovaries over anthers for the embryogenesis induction of several grapevine cultivars. This is the first report of embryogenesis from the cultivars Albariño, Verdejo and Muscat Hamburg as well as transient transformation of Albariño and Tempranillo.     

Somatic embryogenesis and callus production from cotyledon explants of Eastern black walnut

Starting at 8 weeks and continuing until 23 weeks (nut drop) after anthesis,1 m² explants from cotyledons of immature seeds were extracted from Juglans nigra fruits. Explants were placed on Woody Plant Medium with 1 g l^-1 casein hydrolysate and 30 g l^-1 sucrose. The explants remained in light for 4 weeks on primary media containing a 3×3 factorial of 0.05, 0.5, or 5.0 M thidiazuron (TDZ) and 0.1, 1.0, or 10.0 M 2,4-d. Explants were transferred to a secondary medium containing no plant growth regulators and incubated in darkness for 11 weeks. The greatest number of somatic embryos was produced 8, 10, and 12 weeks after anthesis from explants on media with 0.5 or 5.0 M TDZ and 0.1 or 1.0 M 2,4-d. Explants produced the greatest callus volume and dry weight 10, 12, and 14 weeks after anthesis. Throughout the study, callus generally increased with increasing concentrations of both TDZ and 2,4-d.Abbreviations BA 6-benzyladenine - captan 3a,4,7,7a-tetrahydro-2-[(trichloromethyl)thio]-1H-isoindole-1,3(2H)-dione - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - Physan n-alkyl- dimethyl-benzyl ammonium chlorides and n-alkyl-dimethyl-ethylbenzyl ammonium chlorides - TDZ-thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

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.     

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 from Lycopersicon peruvianum leaf mesophyll protoplasts

Summary One to five percent of Lycopersicon peruvianum (L.) Mill. leaf mesophyll protoplasts undergo cell division and concomitant organization to form embryogenic-like structures when cultured in Murashige and Skoog medium (1962) containing 3% sucrose, 9% mannitol, 1.0 mg/l kinetin (K) and 1.0 mg/l naphthalene acetic acid (NAA) at pH 5.6–5.8 (medium A). These embryogenic structures, after passing through developmental stages similar to those observed in zygotic embryogeny, are capable of forming shoots on hormone-free medium A. In medium B, wherein 0.5 mg/l of 2,4-dichlorophenoxyacetic (2,4-D) replaced the hormones (K and NAA), embryogenic structures did not develop. However, callus originating in medium B retained morphogenetic capacity as was evidenced by subsequent shoot regeneration when they were transferred to medium A with K and NAA replaced by 1.0 mg/l zeatin (Z). The potential value of incorporating this regeneration trait into Lycopersicon species and cultivated lines for use in tissue culture programs is discussed.Michigan Agricultural Experiment Station Journal No. 9676     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Somatic embryogenesis and plant regeneration inAzadirachta indica A. Juss

Summary Media for induction of somatic embryogenesis from immature cotyledonary tissues ofAzadirachta indica (Neem) were determined. Callus was initiated on Murashige and Skoog medium supplemented with 0.5 mg·liter⁻¹ of indol-3 acetic acid, 1.0 mg·liter⁻¹ of 6-benzyl amino purine, and 1000 mg·liter⁻¹ of casein hydrolysate. Effect of kinetin was also studied for embryo induction. Carbohydrate source in the form of sucrose and glucose alone and in combination was tested for embryogenic efficiency. Seventy percent embryos showed germination. Healthy plants were potted in sand and soil. Histologic studies confirmed indirect somatic embryogenesis.