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.     

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.     

Nuclear DNA content of somatic and zygotic embryos ofVitis vinifera cv. Grenache noir at the torpedo stage

Summary A flow cytometric analysis and an in situ DNA microspectrophotometric study were made concomitantly to establish why somatic grapevine (Vitis viniferacv. Grenache noir) embryos showed a low level of conversion into plantlets. In somatic embryos at the torpedo stage and in zygotic embryos at the same stage of development, ploidy level, DNA content per 2 C nucleus, and the cell-cycle state of the shoot apical meristem were examined. The frequency distribution histograms of nuclear DNA values were similar in the two types of embryos. At the torpedo stage both types of embryos had a majority of nuclei with 2 C DNA content equal to 1.6pg. In the shoot apices of somatic and zygotic embryos, DNA microspectrophotometry showed preferential blockage of the cell cycle at the G_0–1 stage; however, 20% of somatic embryo shoot apices were blocked at the G_0–2 stage. Analogies between somatic embryos and their zygotic homologues were shown. The genetical and environmental causes of the low level of conversion of grapevine somatic embryos into plantlets are discussed. Our work suggests that the in vitro culture conditions which were used could be incompatible with normal morphogenesis from the torpedo stage.     

Plant recovery from maize somatic embryos subjected to controlled relative humidity dehydration

Summary Maize (Zea mays L.) embryogenic type-II calli were grown on medium containing 0,0.1 μM ABA or 60 g/liter sucrose or both before dehydration of solitary somatic embryos under three relative humidity regimes for up to 6 wk. Viability of dehydrated embryos after 2 wk rehydration was assessed by their ability to produce chlorophyll (greening), roots, coleoptiles, and/or leaves. Only embryos sequentially pretreated with ABA and high sucrose remained viable after 2 wk of dehydration at 70% RH. Up to 34% of the somatic embryos survived 2 wk dehydration at 70% RH, whereas embryos dehydrated at 50 or 90% RH exhibited reduced viability (8.7 and 0.8%, respectively). Approximately 15% of the embryos dehydrated at 70% RH developed into plants, whereas 0.9 and 0% of embryos dehydrated at 50 and 90% RH produced plants. Three percent of maize somatic embryos remained viable after 6 wk of dehydration at 70% RH, and 1.7% developed into plants. Embryo size influenced the ability of maize somatic embryos to survive dehydration. Only embryos greater than 5 mm survived 2 wk dehydration at 70% RH.     

Direct somatic embryogenesis from mature embryos of sandalwood

Plants were regenerated from mature zygotic embryos of sandalwood (Santalum album L.) through direct somatic embryogenesis. Somatic embryos were formed directly without any intervening callus phase on zygotic embryos plated on Murashige and Skoog (MS) medium containing thidiazuron or benzylaminopurine. Individual somatic embryos were then isolated and transferred to MS medium without cytokinin on which they formed secondary embryos in repetitive cycles with or without the addition of indole acetic acid to the medium. Conversion of somatic embryos into plantlets was achieved by isolating somatic embryos with distinct cotyledons and reculturing them onto half-strength MS medium with GA₃ (1.4 M). Recovered plantlets were acclimatised and grown in the greenhouse. This is the first report on in vitro regeneration via direct somatic embryogenesis of sandalwood.     

High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine

Embryogenic suspensions of grapevine (Vitis vinifera L.) were initiated from somatic embryos of `Thompson Seedless' and `Chardonnay'. Suspension cultures consisted of proembryonic masses (PEM) that proliferated without differentiation in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). `Chardonnay' somatic embryos developed fully from PEMs following subculture in medium without 2,4-D; however, somatic embryo development did not advance beyond the heart stage in `Thompson Seedless' suspension cultures. Highly synchronized development of somatic embryos was obtained by inoculating <960-μm PEMs into liquid medium without 2,4-D. Somatic embryos were also produced in large numbers from suspension-derived PEMs of both cultivars on semisolid medium lacking 2,4-D. Somatic embryos matured and regenerated into plants in MS basal medium containing 3% sucrose. Using this method more than 60% of the somatic embryos regenerated plants. More than 90% of the regenerated plants were successfully transferred to the greenhouse. Received: 27 July 1998 / Revision received: 15 October 1998 / Accepted: 27 October 1998     

Desiccated quiescent somatic embryos of orchardgrass for use assynthetic seeds

Summary Somatic embryos of orchardgrass became quiesent when desiccated to 13% water. Twelve percent germinated after 21 d of desiccated storage at 23° C and 4% developed into green plants. During desiccation, embryos decreased in size, became yellowish and brittle, and their outer walls collapsed. Within 15 min after imbibition, they rapidly enlarged and were indistinguishalbe from nondesiccated embryos. These results suggest that somatic embryos may be engineered to function as synthetic seeds for mass propagation. This research represents a collaborative effort between the Agricultural Experiment Stations of the University of Florida and the University of Tennessee. Research was supported in part by the Competitive Research Grants Office of the U.S. Department of Agriculture, grant 82-CRCR-1-1086, awarded to the University of Tennessee. Florida Agricultural Experiment Station Journal Series No. 7173.     

High frequency plant regeneration from Aralia cordata somatic embryos

An efficient method of plant regeneration from Aralia cordatasomatic embryos was developed. Somatic embryos at early stages obtained through inflorescences–derived embryogenic cell suspension cultures were matured in liquid Murashige and Skoog (MS) medium containing various concentrations of abscisic acid (ABA). For plant regeneration, mature cotyledonary embryos were transferred to solid MS basal medium for 6 weeks. Plant regeneration frequency of the embryos matured from heart-shaped embryos was proportional to the concentration of ABA from 0.76 to 3.8 M. The highest frequency (60.7%) was obtained from 3.8 M ABA pretreatment. The survival rate of the plantlets after transfer to plastic pots containing vermiculite in the growth room was 90%. All plants transferred to soil in greenhouse survived. The results indicate that micropropagation procedure can be applied for an efficient mass propagation of Aralia cordata.     

Plantlet regeneration from encapsulated somatic embryos of hybrid Solanum melongena L

High frequency somatic embryogenesis was induced from leaf expiants of F1 hybrid Solanum melongena L. on Murashige and Skoog's medium supplemented with 8.0 mg/1 NAA and 0.1 mg/1 Kn. The somatic embryos were encapsulated in various concentrations (2–6%) of sodium alginate and complexed with calcium chloride (25–100mM): 3% sodium alginate and 75 mM calcium chloride were found to be optimal for encapsulation. The encapsulated somatic embryos were transferred to various conversion media in vitro and in vivo. The frequency of plantlet regeneration varied from 27.0–49.7% in vitro and 2.0–4.5% in vivo.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid     

Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.)

Isolated somatic embryos from petiole-derived callus cultures ofVitis rupestris Scheele have been employed in experiments on genetic transformation. Co-cultivation of somatic embryos during embryogenesis induction withAgrobacterium tumefaciens strain LBA4404, which contains the plasmid pBI121 carrying the neomycin phosphotranspherase and the-glucuronidase genes, produced transformed cellular lines capable of recurrent somatic embryogenesis. Precocious selection for high levels of kanamycin (100 mgl^-1) was an important part of our transformation protocol. Transformed lines still have strong-glucuronidase expression as well as stable insertion of the marker genes after 3 years of in-vitro culture, during which they have maintained their capacity to organize secondary embryos and to regenerate transgenic plants with an agreeable efficiency (13%).     

Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos

Summary For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl⁻¹ (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl⁻¹ (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.     

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     

Direct shoot organogenesis and plant regeneration from leaves of grape (Vitis spp.)

Adventitious shoots developed from in vitro-grown leaves of Vitis vinifera cultivars Cabernet Sauvignon, French Colombard, Grenache, Thompson Seedless (syn. Sultana) and White Riesling, V. rupestris cv. St. George (syn. du Lot) and V. vinifera × rupestris cv. Ganzin 1. Leaf explants less than 15 mm long were excised from nodal cultures and cultured on Murashige and Skoog or Nitsch and Nitsch-based regeneration media with 0, 1, 2 or 4 mgl^-1 6-benzylaminopurine (BAP). Adventitious shoots developed within 4 weeks at the petiolar stub and occasionally from wounded lamina tissues. Shoot organogenesis occurred only on media containing BAP and at a higher frequency with 2 mgl^-1 than with 1 or 4 mgl^-1. On media containing 2 mgl^-1 BAP, 47, 67, 60, and 42%, respectively, of leaf explants of Cabernet Sauvignon, French Colombard, Thompson Seedless, and White Riesling produced adventitious shoots compared to 14, 14, and 29%, respectively, for Grenache, St. George, and Ganzin 1. Solid culture medium was superior to liquid medium and transfer frequency on solid medium did not affect the regeneration frequency. Further shoot growth was promoted by the transfer of regenerating tissues to fresh regeneration medium. More than 80% of explants initially producing adventitious buds exhibited further shoot growth, developing an average of more than 6 shoots each. Shoots rooted easily and the resulting plants appeared morphologically identical to parent vines.     

Direct somatic embryogenesis from axes of mature peanut embryos

Summary Plant regeneration via somatic embryogenesis was obtained in peanut (Arachis hypogaea L.) from axes of mature zygotic embryos. The area of greatest embryogenic activity was a 2-mm region adjacent to and encircling the epicotyl. Somatic embryogenesis was evaluated on Murashige and Skoog media supplemented with a variety of auxin treatments. Maximum production occurred on medium supplemented with 3 mg · liter⁻¹ 4-amino-3,5,6-trichloropicolinic acid. Explant cultures were transferred to half-strength medium supplemented with 1 mg · liter⁻¹ gibberellic acid for somatic embryo germination and early plantlet growth. Plantlets, transferred to soil, were placed in a greenhouse and grown to maturity.     

Transgenic plants of Vitis vinifera cv. Seyval blanc

Leaf discs of grapevine cv. Seyval blanc originating from in vitro cultures were transformed with Agrobacterium tumefaciens strain LBA 4404 harbouring the vector pGJ42 carrying genes for chitinase and RIP (ribosome-inactivating protein) in an attempt to improve fungal resistance. The gene for neomycin phosphotransferase II (nptII) was used as the selectable marker gene. The explants were cocultivated for 2 days with recombinant Agrobacteria and then submitted to selection on NN69 medium containing 100 mg/l kanamycin. Successful regeneration and conversion of transgenic plantlets were obtained. Stable integration of foreign DNA was confirmed by PCR and Southern blot analyses, and protein expression was detected by Western blot. The regenerated transgenic plants were adapted to the greenhouse and showed no evidence of phenotypical alterations. The foreign genes introduced into the transformed plants did not effect the expected improvement in fungal disease resistance under field conditions for the major pests Uncinula necator and Plasmopara viticola.     

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.     

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.     

Direct somatic embryogenesis and plant regeneration from protoplasts of Bupleurum scorzonerifolium Willd

Protolasts of Bupleurum scorzonerifolium were prepared from stem node-derived embryogenic calli with an enzyeme mixture, in which snailase was a necessary component. Follolwing cell wall regeneration protoplasts divided and directly formed somatic embryos which developed into plantlets. The conditions favorable to direct embryo formation were investigated, and the nature of the callus used for protoplast preparation was found to be a critical factor. The osmotic concentration and the composition of the culture medium including the phytohormone combinations were also important.