Somatic embryogenesis from whole flowers,anthers and ovaries of grapevine (<Emphasis Type="Italic">Vitis</Emphasis> spp.)

A novel method for initiating somatic embryogenesis in grapevine, based on immature whole flower culture, is presented. The embryogenic competence of flowers was compared to that of anthers and ovaries, the most widely used explant types, for five grapevine cultivars. Both the genotype and the explant source affected the differentiation of somatic embryos. The highest percentages of embryogenesis were obtained in ovary-derived calli from all cultivars tested with the exception of Brachetto a grappolo lungo. Whole flowers proved to be suitable material for initiating embryogenic cultures for most tested cultivars, and for 110 R, Chardonnay, and Grignolino they gave similar or better results than anthers. Collection of whole flowers from the inflorescence is easier and faster than excision of anthers and ovaries from the flower itself; it can be done without the use of a stereomicroscope and damage to the explant is unlikely. No morphological difference was noted among embryogenic cultures originated from ovaries, flowers, or anthers.     

Detection of somaclonal variants in somatic embryogenesis-regenerated plants of <Emphasis Type="Italic">Vitis vinifera</Emphasis> by flow cytometry and microsatellite markers

Flow cytometry and microsatellite analyses were used to evaluate the trueness-to-type of somatic embryogenesis-regenerated plants from six important Spanish grapevine (Vitis vinifera L.) cultivars. Tetraploid plants were regenerated through somatic embryogenesis from all of the cultivars tested with the exception of ‘Merenzao’. In addition, an octoploid plant was obtained in the cv. ‘Albariño’, and two mixoploids in ‘Torrontés’. The most probable origin of these ploidy variations is somaclonal variation. The cv. ‘Brancellao’ presented significantly more polyploids (28.57%) than any other cultivar, but it must be noted that 50% of the adult field-grown ‘Brancellao’ mother plants analysed were mixoploid. Hence, it is probable that these polyploids originated either from somaclonal variation or by separation of genotypically different cell layers through somatic embryogenesis. Microsatellite analysis of somatic embryogenesis-regenerated plants showed true-to-type varietal genotypes for all plants except six ‘Torrontés’ plants, which showed a mutant allele (231) instead of the normal one (237) at the locus VVMD5. There was not a clear relationship between the occurrence of the observed mutant regenerated plants and the callus induction media composition, the developmental stage of the inflorescences, the type of explant used for starting the cultures or the type of germination (precocious in differentiation medium or normal in germination medium) in any of the cultivars tested, except ‘Torrontés’. The mutant plants described herein have been transplanted to soil for future evaluation of putative phenotypic traits of interest. These mutants can be useful both for breeding programs and for functional genomic approaches aimed at increasing knowledge of the biology of grapevine.     

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.     

Establishment of embryogenic cultures and plant regeneration in the Portuguese cultivar ‘Touriga Nacional’ of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L.

‘Touriga Nacional’ is the most important Portuguese grapevine cultivar used for Port wine, table wine and varietal wine production. In order to obtain a reproducible plant regeneration system that allows the application of biotechnological tools to grapevine breeding, embryogenic cultures were induced from immature flowers of three Touriga Nacional selected clones. Gynoecia and anthers were cultured on Nitsch and Nitsch (Science 163:85–87, 1969) basal medium supplemented with four combinations of the growth regulators 6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetyl-l-aspartic acid (IASP), at 28°C, in the dark. Primary callus, observed on anthers and gynoecia in all media, produced embryogenic callus when cultured on differentiation medium, at 24°C under light. The efficiency on induction of embryogenic callus ranged from 1.2 ± 4.7% to 7.9 ± 13.8% in anthers, and from 17.9 ± 24.9% to 25.3 ± 22.9% in gynoecia. Seven lines of embryogenic cultures were established from the three clones. Multiplication of embryogenic calluses was successfully obtained in maintenance medium, at 26°C, in the dark. These embryogenic calluses produced somatic embryos when subcultured on differentiation medium, under a 16 h photoperiod. Somatic embryos were isolated and cultured on germination medium to achieve conversion which ranged from 35.3 ± 48.5% to 72.7 ± 45.6%. The plantlets obtained were cultured in medium without growth regulators. Secondary embryogenesis was also frequently observed in the hypocotyl-root transition region of somatic embryos. Although some morphological variation occurred between somatic embryos, the regenerated plantlets had a normal phenotype. Maintenance of embryogenic cultures has been achieved since 2002.     

Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation

An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

Somatic embryogenesis from stamen filaments in grapevine (Vitis vinifera L. cv. Mencía): changes in ploidy level and nuclear DNA content

Somatic embryogenesis was induced from stamen filaments and an embryogenic suspension culture was established in the grapevine cultivar Mencía using thidiazuron and 2,4-dichlorophenoxyacetic acid. Four combinations of each growth regulator were assessed for somatic embryo induction in a basal medium containing Nitsch and Nitsch salts and Murashige and Skoog vitamins, and an embryogenic suspension was established in liquid medium containing 1 μM 2,4-dichlorophenoxyacetic acid plus 4.5 μM thidiazuron. By using thidiazuron instead of benzyladenine, induction rates were improved over those previously reported for this cultivar and were relatively high compared with previous results in other cultivars. Three combinations of indole-3-acetic acid and benzyladenine and two inoculum levels were tested in a differentiation medium containing activated charcoal. The size of the inoculum affected the developmental stage of the somatic embryos, whereas the type of growth regulator did not. Both the germination and plant conversion rates were high (87.8% and 88.2%, respectively). An analysis of plant ploidy levels by flow cytometry revealed that 5.6% of the somatic embryo-derived plants were tetraploid. The mean nuclear DNA content of the diploid somatic embryo-derived plants was, on average, 6.7% lower than that of diploid field-grown plants, indicating that this protocol produces low levels of somaclonal variation. The results obtained here indicate that such variations in grapevine can occur both through changes in the ploidy level and by loss of genetic material during somatic embryogenesis.     

Somatic embryogenesis and long term maintenance of embryogenic lines from fox grapes

In the present paper, a method for the induction and long-term maintenance embryogenic cultures for Vitis × Labruscana `Niagara' and `Fredonia' is reported. Embryogenic cultures from these two cultivars were induced in an embryogenesis establishment medium (CIM) from ovaries obtained from flowers 10–14 days pre-anthesis. The embryogenic lines obtained in this experiment have been stably maintained for more than 2 years, through repeated subcultures on a long-term maintenance medium (LTMM) without loss of embryogenic competence. Somatic embryo regeneration and maturation have been successfully achieved after 30 days of cultivating embryogenic cultures in an embryo regeneration medium (EDMM), supplemented with charcoal and polyethylene glycol. The somatic embryos were successfully germinated in two different media, `Fredonia' germination medium (FGM) and `Niagara' germination medium (NGM), and converted into normal looking plants on a conversion medium (CM). This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars

Six commercially important soybean cultivars and one control cultivar were compared for differences in induction-efficiency of somatic embryogenesis, primary embryo yield, and embryo conversion. Cotyledons from immature seeds of similar developmental stage for all soybean cultivars were used for embryo induction. The experiments utilized a Latin square design to exclude the effect of differential lighting and position due to plate location in the growth chamber on the embryogenesis process. Results indicated that the efficiency of embryo induction and yield of primary somatic embryos were genotype-dependent. In contrast, no dependence on genotype was observed for the conversion of embryos to form roots and shoots. The percentage of cotyledons that gave a positive embryogenic response ranged from 26 to 89% for the soybean cultivars tested. The average number of primary globular-stage embryos per responding cotyledon after one month on induction medium ranged from 6 to 13 among the seven cultivars. Conversion frequencies for all genotypes ranged from 27 to 45%.     

Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature

We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries.     

Somatic embryogenesis from immature peach palm inflorescence explants: towards development of an efficient protocol

Various factors affect the induction of somatic embryogenesis in peach palm (Bactris gasipaes Kunth). Among these, both the type and level of auxins had the greatest influence on in vitro responses, although the genotype and the developmental stage of the explants also influenced results. Younger inflorescences were more competent to respond to SE induction than more mature inflorescences and the use of a pre-treatment with 2,4-D (200 μM) in liquid MS culture medium also increased the embryogenic capacity, and diminished the development of flower buds. Higher oxidation rates were observed in explants maintained on 2,4-D-supplemented culture medium, while on 300 μM or 600 μM Picloram and Dicamba lower oxidation rates were observed. The progression from floral meristem to flower bud occurred at high frequency when low concentrations of auxins were used, independent of the type. Higher concentrations of Picloram or Dicamba reduced or even inhibited flower bud development. Picloram also enhanced the embryogenic induction rate more than 2,4-D and Dicamba, and among the concentrations evaluated 300 μM Picloram enhanced induction for both genotypes, with significant differences between genotypes. The best combination of variables used the least mature inflorescence (Infl1) from genotype I with the 2,4-D pre-treatment and 300 μM Picloram to generate 5 embryogenic calli from 18 explants; 26 embryos were obtained on average from each embryogenic callus. From these, eighteen embryos converted to plantlets and six of these survived transfer to the greenhouse.     

High-frequency embryogenesis and plantlet regeneration from isolated microspores of indica rice

We report high-frequency embryogenesis and plantlet development from microspores isolated from anthers of two indica (IR-43, IR-54) and a japonica (T-309) rice cultivars, without prior nutrient preculture of anthers. Pretreatment stress of anthers with mannitol or a sugar-starvation medium, and use of maltose as the carbohydrate source in the microspore culture medium were found to be critical. Co-culture of microspores with rice ovaries was found beneficial but not essential. More than 60% of the microspores of the japonica variety Taipai-309 and 25–45% of the indica cultivars IR-54 and IR-43 showed induction of non-gametophytic development. Consequently, in the best treatments for IR-43 and T-309, more than 500 microspore-derived embryos could be obtained from a single dish (35 mm) containing about 80,000 microspores. Among the indica cultivars, the maximum response was obtained in the basal medium M-019. Plantlet regeneration occurred in about 9% (T-309), 7% (IR-43) and 2% (IR-54) of the transferred embryo-like structures. Received: 6 November 1996 / Revision received: 18 June 1997 / Accepted: 20 August 1997     

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.     

Cytological analysis of early microspore divisions leading to gametic embryo formation in <Emphasis Type="Italic">Quercus suber</Emphasis> L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.     

The frequency of embryogenic pollen grains is not increased by in vitro anther culture in Nicotiana tabacum L.

The numbers of embryogenic (S) grains present in in-situ mature anthers of Nicotiana tabacum L. were compared to the numbers of embryos and plantlets produced in cultured anthers excised at the optimal mitotic stage of development for anther culture. The Feulgen technique of staining embryos caused a considerable loss of grains from cultured anthers but this did not seriously affect the determination of the percentage of embryos present. In no instance did the numbers of embryos produced exceed the maximum number of S grains found, and the distributions of S grain and embryo frequencies in anthers were similar. In rare instances S grains which had undergone the first embryogenic division were observed in situ. The results indicate that all grains capable of embryogenesis are determined during early flower formation and that their number is not increased by in vitro culture.     

Improvements in the production of doubled haploids in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) through isolated microspore culture

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C₁₇ induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F₁ crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named ‘Lanuza’, has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.     

Microspore embryogenesis induced through in vitro anther culture of almond (<Emphasis Type="Italic">Prunus dulcis</Emphasis> Mill.)

Anther culture is one of the most widely used methods to induce gametic embryogenesis. The aim of this investigation was to induce microspore embryogenesis in almond (Prunus dulcis Mill.), through this technique. Anthers were cultured at the vacuolated developmental stage, and seven cultivars, two culture media and two temperature treatments were assessed. Although evidence of the microspore induction was observed in all the genotypes and treatments tested (symmetrical nucleus division and multinucleated structures), calli were produced merely by anthers cultured in the medium P and the regeneration of embryos was detected only in anthers of the cultivars Filippo Ceo, Lauranne and Genco, placed on medium P and subjected to the Control treatment (direct culture at 25?±?1?°C, without the hot thermal shock at 35?±?1?°C for 7 days). Characterization by SSR marker analysis of the embryo genotypes revealed that the regenerants had a single allele for each locus whereas the parent cultivar was heterozygous, indicating their development from haploid microspores. This study reports the evidence of gametic embryogenesis and, particularly, of microspore embryogenesis through in vitro anther culture, in almond, and, for the first time to our knowledge, the production of homozygous embryos.     

Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch)

An efficient and robust protocol to induce embryogenesis in lovage (Levisticum officinale W.D.J. Koch) has been developed. Immature anthers, with most of the microspores at the late uninucleate stage, were used as explants, and embryogenesis was induced in medium with combinations of plant growth regulators including α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). The frequencies of in vitro embryogenesis ranged from 0.42 to 18.25% depending on the combinations of plant growth regulators in the induction medium. Induced globular embryos successfully developed into heart and torpedo-staged embryos. Fresh anther explants produced the highest embryo formation rate (17.75%). Anthers treated at 4?ºC for 3, 5, or 8 d, significantly reduced the embryogenic response (to 3.52–7.85%). More embryos were induced when the sucrose content in the medium was increased from 3 to 6% (w/v), but significantly fewer embryos were produced when sucrose was 8% or more. Nearly 20% of fresh anthers were able to produce embryogenic structures when cultured on Murashige and Skoog medium supplemented with 10.74 μM NAA, 8.80 μM BAP, 9.05 μM 2,4-D, and 6% sucrose. Furthermore, when silver nitrate was added to the embryo induction medium at 90 μM, the frequency of anther browning decreased by 30% and the embryo formation rate increased to 24.75% of anthers cultured. In total, 418 plants were regenerated and cytological analysis confirmed 11 haploid lines from 187 samples randomly selected.     

Plant regeneration from embryogenic callus initiated from immature inflorescences of several high-tannin sorghums

A procedure was established for the induction of regenerable calli from immature inflorescence segments of high-tannin cultivars of sorghum (Sorghum bicolor (L.) Moench). Murashige & Skoog's medium with several components altered was utilized for inducing, maintaining, and regenerating the cultures. Embryogenic calli formed at a frequency of 8–70% depending on the genotype. During a ten-month period, 3600 plants were regenerated from eight genotypes tested. Among the developmental stages of immature inflorescence tested (from differentiation of secondary branch primordia to floret formation) no critical differences were found in potential for callusing, embryogenesis or regeneration. Genotypic differences were observed in pigment production, embryogenic callus formation, shoot differentiation, and in maintenance of regeneration capacity.Abbreviations 2,4-D dichlorophenoxyacetic acid This is Journal Paper Number 11972 from the Purdue University Agricultural Experiment Station     

Modification of cell development in vitro: The effect of colchicine on anther and isolated microspore culture in Brassica napus

In an attempt to discover the biological basis of microspore derived embryogenesis, the effect of the antimicrotubule agent colchicine on anther and free microspore embryogenesis was investigated. The microtubule inhibitor colchicine promoted embryogenesis from cultured anthers, both with regard to the number of anthers responding and the number of embryos being produced per anther. A similar promotional response was also observed with cultured microspores. Although the parameters for cultured anthers and free microspores differed, administration of the drug for a short period immediately prior to pollen mitosis I seems to exert the maximum promotional effect. Of the five cultivars of Brassica napus studied, all responded to colchicine treatment. However, the drug did release more embryogenic potential in poor-responding varieties (i.e. Lirawell and Optima) than in the highest responding variety (Topas). Colchicine also resulted in increased embryogenic response in microspores cultured at lower temperatures.These results are considered in terms of models proposed to explain the switch in microspore development from a gametophytic to a sporophytic pathway. The use ofcolchicine as agent to promote embryogenesis in previously recalcitrant species other than Brassica is also discussed.