Plant Regeneration Through Anther Culture of Three Wild Species of Hordeum (H. murinum, H. marinum and H. bulbosum)

Plant regeneration was achieved through anther culture of three wild species of Hordeum (H. murinum, H. marinum and H, bulbosum). Calli or embryoids were formed from microspores in anthers cultured on a medium containing 6-benzylammopurine (BAP) and ficoll. These calli or embryoids regenerated green or albino shoots and roots after transfer to regeneration media. Green plantlets which developed on regeneration media were transferred to soil where they showed further growth.     

In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.)

A protocol for in vitro induction of tetraploids via colchicine-treated somatic embryos from immature zygotic embryos of diploid grapevine (Vitis vinifera L.) is reported. Embryogenic callus was initiated from immature zygotic embryos cultured on Nitsch and Nitsch (NN) medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was transferred to NN medium containing 1.0 mg/l α-naphthalene acetic acid (NAA) and 0.5 mg/l benzyladenine (BA) to establish somatic embryogenesis. The vigorously growing globular embryos were selected and treated by 0, 10 or 20 mg/l colchicine for 1, 2 or 3 days, and then immediately transferred to NN medium supplemented with 0.03 mg/l NAA and 0.5 mg/l BA, for somatic embryo conversion and plant regeneration. The number of surviving embryos and regenerated plantlets following colchicine treatment decreased with increasing colchicine concentration and treatment time. Among 29 randomly investigated plantlets regenerated from colchicine-treated somatic embryos, five solid tetraploids (2n = 4× = 76) were identified by chromosome counting analysis; all others were diploid (2n = 2× = 38). Ploidy level of plant regenerated was also determined from leaves using flow cytometry. No chimeras with both 2C and 4C nuclei was produced from colchicine-treated somatic embryos. Significant differences in leaf stomata parameters were observed between diploid and induced tetraploid plantlets.     

Callusing and Regeneration of Plantlets via Somatic Embryogenesis from Inflorescence Cultures of Triticum aestivum L.: Role of Genotype and Long-Term Retention of Morphogenic Potential

In the present investigation we have succeeded in obtaining a high frequency of regeneration of plantlets via somatic embryogenesis from callus derived from immature inflorescence explains of Triticum aestivum var, ‘Sonalika’. The explants were cultured on MS medium supplemented with 2,4-D, casein hydrolysate and coconut milk. A large number of embryoids germinated, to form plantlets on the medium when 2, 4-D was omitted altogether or provided at low concentration. Plantlets were transferred to soil under natural environmental conditions and were shown to have the normal chromosome number of 2n = 6x = 42. Experiments with nineteen other varieties show that there is a marked effect of genotype both on initiation of callusing as well as on regeneration. So far ‘Sonahka’ has proved to be the most responsive among varieties tested by us. With callus of the variety ‘Sonalika.’, we also conducted an investigation on long-term retention of regenerative potential. During Song-term culture, for about 12 months, the morphogenic potential gradually diminished and was finally lost, but the regeneration potential could be restored by subculturing at very short intervals.     

Towards development of Al-toxicity tolerant lines in indica rice by exploiting somaclonal variation

Somaclonal variations, induced in vitro, were used to enhance tolerance to aluminium (Al) toxicity in rice. Tolerant plants were developed through in vitro screening of embryogenic calli. The calli were derived from mature seed embryos and cultured on medium stressed with different concentrations of Al₂(SO₄)₃⋅18H₂O. Seed germination, callus induction, plantlet regeneration and callus health declined with increased concentration of Al. At higher Al concentrations, callus health deteriorated drastically with partial to total necrosis. Plantlet regeneration varied largely among varieties and treatments. The variety IR72 produced maximum plantlets among all genotypes tested. An amount of 60 ppm or more Al was highly toxic, which greatly reduced plantlet regeneration from callus. R₀ plantlets were grown under glasshouse. Based on the appearance of bronzing symptoms on leaves, the tolerant R₁ plants were selected. R₁ and R₂ lines derived from putative tolerant somaclones, were evaluated in fiberglass tanks filled with Al-toxic soil. R₃ population was evaluated in the field. A few lines derived from IR72 showed high yield and good plant type. The progenies at R₃ showed normal root growth under stressed environment in sand culture. The study revealed that in vitro screening would be an appropriate alternative to conventional breeding in evolving Al-tolerant lines as observed in case of other abiotic stresses. The technique was useful in creating de novo synthesized Al-tolerance character in rice.     

Influence of culture medium and in vitro conditions on shoot regeneration in Solanum phureja monoploids and fertility of regenerated doubled monoploids

Incorporation of monoploids in practical breeding requires an efficient method of producing homozygous diploids. This study investigated factors for efficient regeneration of doubled monoploid (DM) potato, crossability of DMs and their field performance. Incorporation of silver thiosulphate (STS) in propagation medium for in vitro monoploid plantlets of Solanum phureja did not affect the frequency of cells with diploid(2x) nuclei but increased those with monoploid (1x) and decreased those with 4x nuclei. Higher shoot regeneration was obtained from leaf explants compared with stem explants. Induction of shoots from calluswas affected neither by light during incubation nor by overnight treatment with a benzyl adenine pulse. Female fertility based on seed set after pollination of DMs with heterozygous diploid clones varied among DMs but encouraged their utilization in practical breeding.     

Effect of antiviral chemicals on in vitro regeneration response and production of PLRV-free plants of potato

Potato leaf roll virus (PLRV) is causing serious loss in yield and quality of potatoes. In the present study, the effect of seven antiviral chemicals viz. Acyclovir, 5-Azacytidine, Cytarabine, 5-Bromouracil, Ribavirin, 2-Thiouracil and Zidovudine on regeneration response and production of PLRV-free plants under in vitro conditions is reported. MS medium supplemented with 0.1 mg L^?1 GA₃, 0.1 mg L^?1 NAA and 500 mg L^?1 malt extract was used for regeneration of plantlets from nodal explants. DAS-ELISA and RT-PCR was used for virus indexing of the mother plant and in vitro-regenerated plantlets. Explants of PLRV positive potato plants were cultured on this medium containing different concentrations (5 – 30 mg L^?1) of antiviral chemicals. Shoot regeneration response varied between tested antiviral chemicals and was decreased with increase in concentration of antiviral chemicals from 5 to 30 mg L^?1. Antiviral chemicals at 30 mg L^?1 concentration showed strong inhibitory effect on regeneration response of shoots. In vitro regenerated plantlets tested negative in both ELISA and RT-PCR were only considered as virus free. When regeneration response and number of virus-free plants produced was compared, 2- thiouracil and ribavirin (25 mg L^?1) were found to be effective. 2- thiouracil (25 mg L^?1) gave 38.68% PLRV free plants with 30.55% cultures showing shoot regeneration and ribavirin (25 mg L^?1) gave 39.62% PLRV-free plants with 36.80% cultures showing shoot regeneration. Regeneration response of explants was better on 5-Bromouracil at 30 mg L^?1 concentrations but it was found least effective in production of PLRV-free potato plants.     

Medium, Explant and Genotype Factors Influencing Shoot Regeneration in Oilseed Brassica spp.

The effects of culture media, explants and genotypes on shoot regeneration in oilseed Brassica species were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog medium supplemented with 3 mg l^?1 6‐benzylaminopurine and 0.15 mg l^?1 1‐naphthaleneacetic acid. The addition of 2.5 mg l^?1 AgNO₃ was very beneficial to shoot regeneration in B. napus and Ag₂S₂O₃ (10 mg l^?1) was even superior to AgNO₃ (2.5 mg l^?1). Explant age, explant type and carbon source also significantly affected shoot regeneration. Four‐day‐old seedlings of cotyledonary explants showed the maximum shoot regeneration frequency and number of shoots per explant. Of the four explants – peduncles, hypocotyls, cotyledons and leaf petioles – cotyledons produced the highest shoot regeneration frequency (56.67 %). Four carbon sources – glucose, maltose, starch and sucrose – were compared for their respective effects on shoot regeneration from cotyledonary explants. Sucrose appeared to be the best carbon source for shoot regeneration with the highest shoot regeneration frequency (76.00 %). Considerable variation in shoot regeneration from cotyledonary explants was observed both between and within Brassica species. The shoot regeneration frequency ranged from 10.00 % for cv. R5 (B. rapa) to 83.61 % for cv. N1 (B. napus). Two B. napus, one B. carinata and one B. juncea cultivars exhibited shoot regeneration frequency higher than 70 %. In terms of the number of shoots produced per explant, B. rapa showed the highest variation, ranging from 5.64 for cv. R3 to 1.33 for cv. R5. Normal plantlets were regenerated from all induced shoots and developed normally. The regenerated plants were fertile and identical with the source plants.     

Seed dormancy mechanisms in warm season grass species

Embryogenic protoplasts of Dancy tangerine (Citrus reticulata Blanco) were X-ray irradiated at three doses and electrofused with iodoacetic acid-treated embryogenic protoplasts of Page tangelo (C. reticulata Blanco × C. paradisi Macf.). Shoots could regenerate only from the fusion combination with the lowest irradiation dose, but were recalcitrant to rooting. In vitro grafting was applied to obtain complete plants. Chromosome examination showed that the plants contained mainly diploid and aneuploid cells, together with few tetraploid cells, indicating that they were mixoploids. Random amplified polymorphic DNA analyses with 10-mer arbitrary primers confirmed the plants as true somatic hybrids. This is the first report on regeneration of mixoploid hybrid plants via protoplast asymmetric fusion in Citrus. Negative effects of ionizing irradiation on regeneration of embryoids and plantlets and possible agronomic interest of the mixoploid plants are also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

An efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation protocol for black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.) using embryogenic mass as explant

A protocol was developed for an efficient Agrobactertium-mediated transformation of black pepper plants through somatic embryogenesis. Embryogenic mass derived from primary somatic embryos that were obtained from the micropylar region of mature germinating seeds of black pepper was found to be the ideal target tissue for transformation. Genetic fidelity test of embryogenic mass-derived plantlets by RAPD using 23 random primers revealed no genetic variation among the progenies and the parent plant. Among the antibiotics used for selection of transformants, cefotaxime at 100 μg mL⁻¹ was found to be optimum to control Agrobacterium besides its ability to promote somatic embryo proliferation. In the case of kanamycin, a step-wise increase in concentration from 25 to 50 and then to 100 μg mL⁻¹ were found to be optimum. Embryogenic mass co-cultivated with Agrobacterium carrying the β-glucuronidase (GUS) reporter gene were cultured on plant growth regulator-free Schenk and Hildebrandt (SH) medium and transformants were selected in selection medium containing cefotaxime and step-wise increase in kanamycin concentration. The transient GUS gene expression was determined histochemically. Transformants that survived in the selection medium were hardened in the greenhouse. An average of nine hardened putative plantlets was obtained per gram of embryogenic mass. The presence of transgene in these plantlets was assayed by PCR, dot blot, and Southern blot hybridization. Results presented demonstrated for the first time an efficient transformation and regeneration of black pepper without the use of growth regulators. This simple efficient procedure would allow transformation of black pepper with genes of desirable characters.     

Culture of unpollinated ovules,ovaries, and flower buds in some species of the genus Allium and haploid induction via gynogenesis in onion (Allium cepa L.)

Summary Induction of haploid plants is of great importance for breeding purposes because of the possibility to obtain from haploids homozygous material by artificial chromosome doubling in relatively short times. The present study reports the first evidence of successfull haploid induction in onion. Isolated ovules, ovaries, or whole flower buds of different Allium species were cultured on BDS agar medium. Testa browning in the ovules and an extensive growth of the latter were observed. In cultures of ovaries and flower buds, development of callus and subsequent regeneration of plantlets from the region of the nectaries were observed. In leek, sometimes supernumerary flower organs like ovules were formed in this callus. In onion (Allium cepa L.), plantlets developed from the ovules in all culture methods. Chromosome numbers of these plantlets were counted in root tip squash preparations. They were found to be haploid. Haploid plants were significantly smaller than diploid ones. They were transferred to soil and developed until bulb formation. Because of their importance for breeding, haploid plants obtained by gynogenesis are further stored in vitro.     

Phenylacetic acid stimulation of direct shoot formation in anther and somatic tissue cultures of rice (Oryza saliva L.)

The effect of phenylacetic acid (PAA) on rice (Oryza saliva L.) anther culture was investigated with six genotypes, using 2,4-D as control. In the two-step culture protocol, replacing 2, 4-D with PAA in the induction medium did not influence callus induction but significantly improved the shoot differentiation from callus, particularly in the indica cultivar Teqing. The anther-derived calli of all genotypes regenerated shoots directly on the callus induction medium containing PAA. Most of the directly-regenerated plantlets had well-developed root systems and were therefore readily transplanted into soil. The improved shoot differentiation potential and the frequency of direct regeneration depended on genotype, basal medium and PAA concentration. The one-step green shoot regeneration frequencies obtained were 1.98% with the indica cultivar ‘129’, 1.5% with the indica × japonica hybrid ‘Teqing/02428’ (F₁), and 1.98% with the indica × indica hybrid ‘Waiyin 2/C.B.’ (F₁). The PAA-based one-step method was most effective on the anther culture of indica genotypes. Three DH populations have been constructed from hybrids (F₁) via one-step culture. PAA also enhanced the one-step plantlet formation in rice somatic tissue culture.     

Continuous Plant Regeneration from Established Embryogenic Cell Suspension Cultures of Italian Ryegrass and Tall Fescue

The suitability of different protocols was compared for entire plant regeneration by somatic embryogenesis, of the forage plants Lolium multiflorum Lam. (Italian ryegrass) and Festuca arundinacea Schreb. (tall fescue). In the first protocol, miniature embryos were used as starting material, while mature seeds were retained in the other two. Whichever the considered protocol, undifferentiated calli were produced on Murashige and Skoog MS medium supplemented with 2,4-D. The calli were subcultured in the dark on solid MS agar medium, containing 5 mg/1 2,4-D (protocol 2) or on solid MS medium followed by transfer to a rotated liquid MS medium with 2 mg/1 2,4-D (protocol 1). In these conditions, induction of somatic embryogenesis occurred, and whole plants were regenerated during a limited lapse of time, upon transfer in the light, to MS medium supplemented with BAP but devoid of 2,4-D. The simultaneous elimination of 2,4-D and transfer to light appeared essential for full regeneration of the plants. Using this characteristic, an additional step was added to a new protocol (protocol 3) in which microcalli, cultured on liquid MS medium containing 5 mg/1 2,4-D, were transferred to the same medium with 2 mg/1 2,4-D, in the dark. In these conditions, the suspensions kept their embryogenic potential for months. In all cases, plantlets were successfully transferred into the soil. An evaluation of the somaclonal variation potential of the plants issued from each protocol is now underway.     

Callus Production and Plant Regeneration from Mature Embryos of Poa pratensis L.

Plant regeneration from callus cultures may provide a source ot somaclonal variation for the improvement ot the apomictic grass Poa pratensis L. It is first necessary to be able to induce callus and regenerate plants in this species at a high frequency. Variation was observed between 50 cutivars of Poa pratensis for callus induction and plant regeneration. Using the cultivars ‘Merion’ and ‘Victa’, three basal media were tested along with various media additives. Murashige and Skoog's basal medium with 0.2 mg 1^?1 2,4-dichlorophenoxyacetic acid, 0.1 mg 1^?1 6-benzylamanopurine, 100 mg 1^?1 casein hydrolysate and 25 g 1^?1 sucrose is considered to be a good medium for callus growth and plant regeneration. Embryo-like structures were observed in the callus of some cultivars but plant regeneration appeared to be predominantly from shoot meristems on the callus surface. The majority of regenerated shoots were green, but chlorophyll deficient shoots were obtained from media containing coconut milk. Green plantlets could be transferred to soil without difficulty.     

Regeneration in sugarcane via somatic embryogenesis and genomic instability in regenerated plants

In the present study, embryogenic calli of sugarcane variety BL4 were induced using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in different concentrations and combinations. In contrast to earlier studies, embryogenic callus sectors were identified and isolated microscopically within 1–2 weeks. Subsequently, 51 media formulations were used for regeneration of proliferated embryogenic callus, using MS medium supplemented with three different cytokinins [kinetin, 6-Benzylamino purine (BAP), and thidiazuron (TDZ)] and auxins (IAA/NAA and IBA) in different combination and concentrations. After acclimatization, the genomic DNA of regenerated plants was studied to explore the insertion polymorphism of transposable elements in order to ascertain the variation among somaclones. Though low concentration of kinetin with 2,4-D was found supportive to embryogenic callus development, the highest number of regenerated plantlets was observed using BAP (1 μM), however the plantlets had very low fresh weight (2.2 g). Conversely, TDZ alone supported a significant increase in the number of plantlets regenerated (38–40) with higher fresh weight. The somaclones generated during this study showed considerable positional polymorphism of activator-like transposable elements possibly due to the stress associated with in vitro culture. This study provides a procedure to produce regenerated sugarcane plants from embryogenic callus in a relatively short time.     

Plant regeneration from callus culture in potato

Summary A procedure for plant regeneration from callus culture of potato, Solanum tuberosum L. is described. Calli were induced from 1–2 mm long shoot apices of potato cultivars Cara and A25/19 on half-strength Murashige and Skoog's medium (half-MS) supplemented with 3.2 mg IAA (indole-3-acetic acid), 1.0 mg kinetin (6-furfurylamino)purine], and 0.5 mg 2,4-D [2,4-dichlorophenoxy)acetic acid]/1. Sixty percent explants produced nodular calli on this medium within 30 days. Calli differentiated into shoot-primordia when subcultured on half-MS medium supplemented with 0.5 mg 2,4-D and 1.0 mg zeatin [6-(4-hydroxy-3-methybut-2 enylamino)amino purine]/1. Differentiated calli on half-MS medium without growth hormones produced complete plantlets which were cloned on the same medium and transferred into soil.     

Somatic cell genetic studies inBrassica species. II. Production of androgenetic haploid plants inBrassica nigra (L.)Koch

Summary Callus growth and its subsequent regeneration into complete plantlets was achieved from in vitro cultured anthers ofBrassica nigra (L.)Koch. Callus was induced on a modified N6 medium containing trace elements, organics of B5 medium and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Morphogenesis of callus in the form of shoots on MS medium containing indole-3-acetic acid (IAA) and N⁶-benzyladenine (BA) 0.5 mg/l each and embryoids on MS medium containing 0.5–1.0 mg/l IAA and 3.0–5.0 mg/l BA could be accomplished. Chromosomal analysis revealed presence of 41% haploids (n=8) amongst the regenerated plants.     

Induction of variability in chimeric Aster cordifolius `White Elegans' through somaclonal variation

A protocol was developed for plant regeneration from leaf explants (laminaand petiole) of Aster cordifolius cultivar `White Elegans', which ischimeric for chromosome number and the ligulate flower colour. Explantswere cultured on MS or Gamborg's B5 medium in presence of IAA, 2,4-Dand BAP, alone or in combination. The highest frequency of shootregeneration was obtained on B5 medium supplemented with 2,4-D(0.09 mg l^-1) and BAP (1.8 mg l^-1). After the invitro rooting stage and acclimatization, 90% of plantlets survived in thegreenhouse. The regeneration process occurred by indirect organogenesisas shown by cyto-histology. The regenerants maintained the originalchimerism for chromosome number. Pink flowers instead of theexpected white ones were obtained in all regenerants. Ahypothesis for the uniform colour change is discussed. Variations weredetected in the regenerant generation (R₀) for quantitative characterssuch as capitulum and disc diameters, and number of ligulate flowers. SinceAster can be vegetatively propagated, selection of variants in R₀can provide interesting material for breeding purposes.     

Plant Regeneration and Genetic Variability from Tissue Cultures of Sunflower (Helianthus annuus L.)

Adventitious buds were induced from in vitro culture of sunflower (Helianthus annuus L.) cotyledons. Four inbred lines (G1, G2, G3 and HA89), an open pollinated variety (‘Argentario’) and two hybrids with specific genetic markers were used. Cotyledons were cultured in vitro on MS medium (Murashlge and Skoog 1962) containing various concentrations of kinetin and indole 3-acetic acid (IAA). The quantitative interactions between auxin and cytokinin, the age of the cotyledons and the 2,3,5-triiodobenzoic acid (TIBA) treatments have been found to influence shoot regeneration. The plantlets, after rooting, were successfully established on soil. Qualitative variation was noted in self-pollinated progeny of plants regenerated from culture of two inbreds. Chimerism in regenerated plants was indicated by sectoring of the genetic markers. Some culture-induced variant phenotypes were similar to known spontaneous mutation in sunflower but others have been not yet described. Preliminary data indicate that most of them may have single-gene recessive inheritance.     

Callus formation and plantlet regeneration from immature Triticum durum Desf. embryos

Summary Experiments upon in vitro culture of immature durum wheat embryos, harvested at different growth stages, were made in two consecutive years. Callus formation and plantlet regeneration were obtained. The ability to form callus and the degree of morphogenetic processes varied with the different hormonal treatments used and with the age of the embryos. In the first year the best response for callus growth was observed with 2,4-D 2 mg l^-1 plus adenine 50 mg l^-1 or 2,4-D 5 mg l^-1 alone in the more mature embryos (15 and 20 days after anthesis). On the contrary, NAA 5 mg l^-1 had a greater shoot regeneration effect. In the next year, at all 2,4-D concentrations and for the two different ages of the embryos tested, all embryos formed callus. Regeneration of plantlets was obtained in higher percentage in calli originated from the more developed embryos. The effect of changed media upon plantlet regeneration was studied after callus transplant.Investigation by cytophotometry and chromosome counts on different calli showed, practically in all cells, a diploid condition. A histological analysis demonstrated embryogenic somatic characteristics in many samples of callus. The pattern of organogenesis seemed to be via adventitious bud formation but structures resembling embryoids were also observed in the callus.     

Direct as well as indirect somatic embryogenesis from immature (unemerged) inflorescence of a minor millet Paspalum scrobiculatum L.

Somatic embryos differentiated directly on the rachis of immature inflorescences of Paspalum scrobiculatum L. cv. PSC 1 on culture to MS or N₆ medium supplemented with different concentrations (4.5–22.5 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D). Direct embryogenesis on the rachis of inflorescence explants forms the first instance in graminaceous plants. Highest frequency of direct embryogenesis (34%and 30% cultures, respectively) was possible on N₆ medium supplemented with 4.5 μM of 2,4-D and MS medium fortified with9.0 μM of 2,4-D. Other tissues of the explant, floral-primordia, only after an initial phase of callusing differentiated into somatic embryos; indirect embryogenesis. Somatic embryogenesis, direct as well as indirect, was resolved by scanning electron microscopy. The somatic embryos germinated and developed into plantlets on regeneration medium. Interestingly, one week incubation of somatic embryos on activated charcoal (0.5%) fortified basal medium, supported high potential for ‘germination’ on transfer to charcoal-free basal medium. This beneficial effect of activated charcoal on regeneration of somatic embryos into plantlets is the first record in the Gramineae. This revised version was published online in July 2006 with corrections to the Cover Date.