Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)

Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l⁻¹ agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l⁻¹ (22.8 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l⁻¹ (45.6 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l⁻¹ (1.1. mM) IBA solution.     

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.     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.     

Recovery and regeneration of embryogenic cultures from female flowers of False Horn Plantain

Somatic embryogenesis from immature male flowers in Musa is only suitable for genotypes with a male bud. Six friable embryogenic cultures were obtained from 28 cultured buds of female flowers of the AAB False Horn Plantains, ‘Curraré’ and ‘Curraré Enano’. Embryogenic suspensions were established from these embryogenic cultures. Somatic embryogenesis was demonstrated histologicaly. Regeneration of plants was obtained either from somatic embryos directly isolated from embryogenic cultures or from suspensions after plating on a semi-solid medium. This study demonstrates that somatic embryogenesis from immature flowers is suitable for genotypes of Musa with or without male buds. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Screening of a broad range of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) germplasm for <Emphasis Type="Italic">in vitro</Emphasis> rapid plant regeneration and development of an early prediction system

Rice has emerged as a model monocot for studies in agriculture and biotechnology due to its relatively small genome and a ready accessibility to plant material. Tissue culture is one of the tools required for genetic transformation and some breeding programs, and the selection of high-frequency regenerator types is essential for success in these technologies. Thirty-three rice entries with agricultural and biotechnological characteristics of interest were screened with the aim to identify the best regenerators. Entries that exhibited between 50% and 90% regeneration frequencies include ‘Taipei-309,’ ‘Super Dwarf,’ ‘Norin’ (japonica types), PI 312777, ‘Ali Combo’ (indica types), ‘STG-S,’ and ‘LA3’ (red rice types). One third of the entries tested were at least two times better at regeneration than the often-cited regenerator ‘Nipponbare.’ Those entries showing at least 85% frequency of greening or somatic embryo formation at 15 or 30 d on regeneration medium ultimately produced whole plants after 45 d on regeneration medium at high frequency (at least 40%); those entries not reaching the 85% threshold of greening by Days 15 or 30 exhibited moderate (15–40%) to low (less than 10%) frequency of whole plant regeneration. This greening response suggests the means for an early prediction system for identification of useful rice regenerator lines, which would be beneficial for high-throughput screening of germplasm as well as for decreasing the time and cost of in vitro culture.     

Effect of bialaphos and phosphinothricin on plant regeneration from long- and short-term callus cultures of Gladiolus

Summary Callus was initiated from in vitro-grown plants of Gladiolus cultivars ‘Jenny Lee’ and ‘Florida Flame.’ The age of callus used for regeneration of plants was either 9 mo. old or 8 yr old from ‘Jenny Lee,’ and 4 mo. old from ‘Florida Flame.’ Regeneration medium consisted of Murashige and Skoog’s basal salts medium supplemented with 2.0 mg/l (9.3 μM) kinetin. This medium was supplemented with various concentrations of either bialaphos (Meiji Seika, Tokyo, Japan) or phosphinothricin (Hoechst-Roussell, Frankfurt, Germany). Bialaphos was more effective than phosphinothricin at stimulating plant regeneration. Plants regenerated from 8-yr-old callus of ‘Jenny Lee’ only when the regeneration medium was supplemented with 0.10 mg/l bialaphos. A bialaphos concentration of 0.01 mg/l stimulated regeneration from 9-mo.-old callus of cultivar ‘Jenny Lee’ and 4-mo.-old callus of ‘Florida Flame.’     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

Efficient and rapid plant regeneration of oil palm zygotic embryos cv. ‘Tenera’ through somatic embryogenesis

An efficient and rapid plant regeneration system through somatic embryogenesis was developed using 13-week-old zygotic embryos of oil palm (Elaeis guineensis Jacq.) cv. ‘Tenera’. Zygotic embryos were cultured on MS and N6 media supplemented with 2.0 mg L⁻¹ picloram, 2,4-D and dicamba. The highest embryogenic callus formation (32%) was observed on N6 medium with 2,4-D after 3 month culture on callus induction medium. Somatic embryos were continuously formed from nodular calli on embryo maturation medium [N6 + 0.1 mg L⁻¹ 2,4-D, 0.16 g L⁻¹ putrescine, 0.5 g L⁻¹ casein amino acids and 2.0 g L⁻¹ activated charcoal(AC)] for 3–5 months. Histological analysis confirmed that embryo development occurred via somatic embryogenesis. For plant regeneration, modified N6 medium (MN6) with AC (0.5 g L⁻¹) without growth regulators, induced both shoot and root formation simultaneously with the highest regeneration rate of 56%. This combined shoot and root induction protocol shortened the culture time to 9–12 months. Furthermore, after acclimatization, more than 85% of transferred plants from our protocol developed successfully in the soil.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of <Emphasis Type="Italic">Melia azedarach</Emphasis> (Meliaceae)

Summary A procedure for the regeneration of ‘paradise tree’ (Melia azedarach, Meliaceae) plants from immature zygotic embryos via somatic embryogenesis was developed. Somatic embryos were induced from explants cultured on Murashige and Skoog medium supplemented with 0.45, 4.54, or 13.62 μM thidiazuron. Histological examination revealed that somatic embryos were induced directly from the explants. Further development of somatic embryos was accomplished with Murashige and Skoog medium at quarter-strength with 3% sucrose. A large number of plants were regenerated from somatic embryos and successfully established in soil in a greenhouse. These plants are morphologically similar to those of seed-derived plants. This system may be beneficial for mass propagation as well as for genetic manipulation of the ‘paradise tree’.     

Effects of different amino acids on somatic embryogenesis of strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch.)

This study was conducted to optimize different types and concentrations of amino acids on somatic embryogenesis induction, development and maturation of leaf explants in strawberry cultivars (‘Camarosa’, ‘Paros’ and ‘Kurdistan’). Calli derived from leaf sections were transferred onto MS medium with 1.0 mg/l 2,4-d + 0.5 mg/l BAP supplemented with 0.0, 50, 100, 150 or 200 mg/l concentrations of proline, alanine and glutamine. Stimulation of embryogenesis and embryo development was strictly dependent on the type and concentration of amino acid in the medium. Proline (100 mg/l) was much more effective than glutamine and alanine, on induction and development of somatic embryogenesis in all cultivars. Cultures grown on amino acid-free medium attained lower somatic embryos than cultures grown on amino acid treated medium. Low concentrations (50 mg/l) and high concentrations (200 mg/l) of amino acids tested were inefficient for embryogenesis induction as well as for somatic embryos development.     

Production,storability, and regeneration of shoot tips of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) encapsulated in calcium alginate hollow beads

Summary Shoot tips, of four potato cultivars (Désirée, Genet, Tigoni, and Tomensa), 3–4 mm in size, were precultured for 2 d on Murashige and Skoog (MS) solid medium, then encapsulated in calcium alginate to produce hollow bead synthetic seed capsules averaging 0.78 cm in diameter. Regeneration and ‘regrowth’ were tested on MS solid medium and on soil in the greenhouse, respectively. The encapsulated shoot tips were stored at 4 and 10°C for up to 390 d. For all cultivars, the encapsulated shoot tips stored at both temperatures for 180 d and at 4°C,for 270 d, 100% regeneration on MS solid medium was recorded. After 360 d in storage at 4°C, 70.8% (Tigoni), 66.7% (Genet), 58.3% (Désirée), and 51.5% (Tomensa) regeneration was recorded on MS medium, reducing to 15% (Tigoni), 25% (Genet), 10% (Désirée), and 0% (Tomensa) regeneration after 390 d in storage. ‘Regrowth’ of 93–100% was recorded for non-stored encapsulated shoot tips, directly transferred on soil in the greenhouse after a 2 wk preculture on MS solid medium with an added fungicide (carbendazim) in the encapsulating gel. The ‘regrown’ shoot tips produced plants showing normal development. The results presented here demonstrate that hollow bead synthetic seed capsules are an alternative propagating method for potato seed production.     

In-vitro regeneration of olive tree by somatic embryogenesis

We induced somatic embryogenesis from the cotyledon segments ofOlea europaea (L) cvs. ‘Chetoui’, ‘Chemleli’, and ‘Arbequina’. Calli were established from all three cultvars on OMc media supplemented with IBA and 2i-R The greatest success was obtained with media that contained zero or low concentrations of growth regulators. High levels of hormones (i.e.,>0.5 mgL^-1 IBA and 2i-P) inhibited embryogenesis. Embryos at different maturation stages were observed with continuously proliferating secondary embryogenesis. Abnormally shaped embryos and teratoma were also noted. Four weeks was the optimal incubation period for inducing embryogenesis on the auxin-containing medium. In addition, 30 to 40 gL^-1 sucrose was more effective than glucose in stimulating the growth and maturation of somatic embryos. Embryogeic efficiency was also higher when multivariate combinations of nitrogen sources (inorganic and organic nitrogen forms) were used. The plantlets that were derived from our germinating somatic embryos were similar to those obtained from axillary buds.     

Postulation of resistance genes to barley diseases in heterogeneous varieties

Resistance to causal agents of diseases is an important varietal characteristic that influences the management practice of crop plants and thus production costs of commodities. At present, almost all European barley varieties possess at least one major gene for resistance to powdery mildew. After hybridizing selected parental varieties, resistance genes often segregate in subsequent generations and, therefore, some varieties comprise lines that differ in the number or combinations of resistance genes. The objective of this research was to describe the various methods available for postulating resistance genes to pathogens in heterogeneous varieties using resistance to powdery mildew of barley as an example. Four spring barleys (‘Orbit’, ‘Malva’, ‘Tocada’ and CLE 233), and a six-row variety of winter barley, F 12872, were screened. For postulating resistance genes, several testing procedures and many Blumeria graminis f.sp. hordei isolates were used. Minimum amounts of seed were determined and different methods of obtaining homogeneous seed samples from heterogeneous varieties were compared. It was found that ‘Orbit’ and ‘Malva’ are composed of three and ‘Tocada’, CLE 233 and F 12872 of two lines with different resistances to powdery mildew. Problems of postulating resistance genes in heterogeneous varieties and the advantages of testing leaf segments instead of young plants are discussed.     

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.     

Development of STS markers closely linked to the Ppd-H1 photoperiod response gene of barley (Hordeum vulgare L.)

A BC₂ population of 353 plants segregating for the Ppd-H1 photoperiod response gene was developed from a cross between the winter barley ’Igri’ and the spring barley ’Triumph.’ Bulk segregant analysis identified six AFLP markers closely linked to the Ppd–H1 gene and three strongly amplified AFLP bands that mapped 0.8-cM distal, 0.6-cM proximal and 2.3-cm proximal to Ppd-H1 were cloned and sequenced. Southern-blot analysis showed that the cloned fragments were single-copy sequences in ’Igri’, the variety from which they were derived. Two of the sequences were absent from ’Triumph’ while the third detected a single-copy sequence. The cloned fragments were used to design specific sequence tagged site (STS) primer pairs to assist in the construction of a high-resolution map of the Ppd-H1 region. Received: 22 March 2000 / Accepted: 10 April 2000     

Ethylene influences green plant regeneration from barley callus

The plant hormone ethylene is involved in numerous plant processes including in vitro growth and regeneration. Manipulating ethylene in vitro may be useful for increasing plant regeneration from cultured cells. As part of ongoing efforts to improve plant regeneration from barley (Hordeum vulgare L.), we investigated ethylene emanation using our improved system and investigated methods of manipulating ethylene to increase regeneration. In vitro assays of regeneration from six cultivars, involving 10 weeks of callus initiation and proliferation followed by 8 weeks of plant regeneration, showed a correlation between regeneration and ethylene production: ethylene production was highest from ‘Golden Promise’, the best regenerator, and lowest from ‘Morex’ and ‘DH-20’, the poorest regenerators. Increasing ethylene production by addition of 1-aminocyclopropane 1-carboxylic acid (ACC) during weeks 8–10 increased regeneration from Morex. In contrast, adding ACC to Golden Promise cultures during any of the tissue culture steps reduced regeneration, suggesting that Golden Promise may produce more ethylene than needed for maximum regeneration rates. Blocking ethylene action with silver nitrate during weeks 5–10 almost doubled the regeneration from Morex and increased the Golden Promise regeneration 1.5-fold. Silver nitrate treatment of Golden Promise cultures during weeks 8–14 more than doubled the green plant regeneration. These results indicate that differential ethylene production is related to regeneration in the improved barley tissue culture system. Specific manipulations of ethylene were identified that can be used to increase the green plant regeneration from barley cultivars. The timing of ethylene action appears to be critical for maximum regeneration.     

Highly efficient embryogenesis and plant regeneration of tall fescue (Festuca arundinacea Schreb.) from mature seed-derived calli

Summary We report a protocol for efficient plant regeneration of four tall fescue (Festuca arundinacea Schreb.) cultivars (‘Surpro’, ‘Coronado’, ‘Summer Lawn’, and ‘Fawn’) via somatic embryogenesis. Calli were initiated from mature seeds grown on modified Murashige and Skoog (MMS) medium supplemented with 7.0mgl⁻¹ (31.7μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mgl⁻¹ (0.23 μM) kinetin (Kin). Calli were maintained and proliferated by subculture at monthly intervals on MMS medium containing 4.5 mgl⁻¹ (20.4 μM) 2,4-D and 0.2mgl⁻¹ (0.9 μM) Kin. Somatic embryos (SE) were induced from seed-derived calli on SE-induction medium (MMS supplemented with 2.0 mgl⁻¹ 2,4-D and 0.2mgl⁻¹ Kin). Plantlets were regenerated from somatic embryogenic calli grown on modified SH medium supplemented with 2 mgl⁻¹ Kin. Using this optimized protocol, 78.6–82.3% of mature seeds of all four cultivars produced SE clusters, of which 93.5–95.3% regenerated into plants within 10 wk. The regenerants showed no phenotypic abnormalities.     

Efficient regeneration of fertile barley plants from callus cultures of several Nordic cultivars

The regeneration potential of three major Estonian barley cultivars was tested and compared to that of the Finnish cultivar Kymppi. Two different regeneration systems were used. The first was characterized by the high maltose concentration (60 g l(-1)) and by the use of 2,4D together with two different combinations of amino acids in the callus induction medium followed by the regeneration medium containing BAP (2 mg l(-1)) and 2,4D (0.2 mg l(-1)). The second exploited callus induction medium that contained Dicamba, lower concentrations of maltose (30 g l(-1)) and higher concentrations of myo-inositol and thiamine and different set of amino acids and regeneration medium that contained higher concentrations of Cu2+ and inorganic nitrogen accompanied by lower concentrations of NH4+ and BAP, when compared to the first regeneration system. The second regeneration system used produced significantly higher rates of callus induction, callus growth and regeneration of plantlets. However, it yielded also many albino plants (up to 51%), whereas the first regeneration system used did not produce practically any albino plants. No major genotype-dependent differences were observed in comparison between two regeneration systems - in both systems higher regeneration potential of Anni, Elo and Kymppi contradicted to the low regeneration potential of Teele. It is concluded that the continuous somatic embryogenesis on the regeneration medium allows the regeneration of many plants from the same callus over long periods of time and makes available highly efficient regeneration protocols for Estonian and Finnish barley cultivars.     

Somatic embryogenesis from mesocotyl and leaf-base segments of <Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L., a minor millet

Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N₆) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N₆ medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N₆ regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N₆ medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.