Plant regeneration from callus of Puccinellia distans (L.) Parl

Callus was induced from seeds of Puccinellia distans (L.) Parl. on MS medium supplemented with 2 mgl^-1 2,4-dichlorophenoxyacetic acid and 0.5 mgl^-1 kinetin. Morphogenesis initiation was achieved during subculture on medium containing 0.1 mgl^-1 2,4-D. From the point of morphogenetic capacity, 3 types of callus were selected. High frequency of plant regeneration was obtained by selection of embryogenic type of callus, and culture on N₆ medium and N₆ medium supplemented with kinetin (5–10 mgl^-1), or kinetin (2 mgl^-1) and IAA (0.5 mgl^-1). A high ratio of albinos among regenerants was observed.     

Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted β-glucuronidase gene in regenerants from isolated protoplasts

Cotyledonary expiants of ginseng zygotic embryos were cocultured with Agrobacterium tumefadens strain LBA4404 harboring the binary vector pBI121 for 48 h and transferred onto MS medium supplemented with 1 mgl^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mgl^–1 kinetin, and 100 mgl^–1 kanamycin. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary expiants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto medium containing 1 mgl^–1 each of 6-benzyladenine (BA) and gibberellic acid, most of them developed into plantlets. Southern analysis confirmed that the -glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants. Protoplasts were enzymatically isolated from transformed somatic embryo segments and cultured in liquid medium containing 60 gl^–1 myo-inositol, 1 mgl^–1 2,4-D, 0.5 mgl^–1 BA, and 0.5 mgl^–1 kinetin. Plants were regenerated from protoplasts via somatic embryogenesis. The polymerase chain reaction method revealed that 92% of the regenerants retained the GUS gene. When treated with X-glucuronide, 78% of the regenerants showed a GUS-positive response. The overall results indicate that the transgene is stably transmitted during somatic ontogeny and stably expressed in most the regenerants, whereas it may be deleted or impaired in some portion of them.Abbreviations BA 6-benzyladenine; 2,4-D,2,4-dichlorophenoxyacetic acid - DIG digoxigenine - GA₃ gibberellic acid - X-gluc X-glucuronide - GUS -glucuronidase - MS Murashige and Skoog (1962)     

Salt tolerance improvement in some wheat cultivars after application of in vitro selection pressure

Somatic embryogenesis through callus initiation has been quantified under salt stress conditions for 8 wheat cultivars currently cultivated in Morocco. The cultivars were classed according to the mean number of somatic embryos formed per immature embryo half and regenerated plants per 100 explants under saline conditions. Regenerated plants from control callus (R_0–0) and callus initiated on 10 g l^–1 NaCl (R_0–10) did not show significant differences concerning plant height, spike length and grain number per ear but, the R₀ plants remained less developed than parent plants. When watered with a solution containing more than 20 g l^–1 NaCl, the seeds of cultivar Te derived from R_0–10 regenerated plants exhibited the best elongation of roots and coleoptiles. Furthermore, a chlorophyll fluorescence test showed a clear improvement in salt tolerance of R_0–10 plants at four to five-leaf stage, compared to R_0–0 plants. It is concluded that plant regeneration from callus initiated on high NaCl levels may be a valid method of selection for salt tolerance.     

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris

Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl^–1) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl^–1 indole-3-acetic acid or 0.1 mgl^–1 indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl^–1) with indole-3-butyric acid (0.1 mgl^–1) was added to NN medium supplemented with casein hydrolysate (250 mgl^–1). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NN Nitsch and Nitsch (1969) - NOA 2-naphthoxyacetic acid     

Shoot regeneration of hybrid seed geranium (Pelargonium x hortorum) and regal geranium (Pelargonium x domesticum) from primary callus cultures

Procedures have been developed that increase the rate of shoot regeneration of hybrid seed geranium from month-old primary callus cultures. Hybrid geranium callus tissue covered with green nodular structures was initiated by placing shoot tip explants on solidified Murashige & Skoog medium (MS) supplemented with 2.0 mgl^-1 zeatin and 1.9 mgl^-1 indoleacetic acid. Hybrids Red Orbit, White Orbit and Scarlet Orbit were shown to produce 5–50 shoot primordia per explant when callus was initiated on this medium. Regal geranium callus was initiated by placing leaf explants on MS medium supplemented with 2.0 mgl^-1 6-benzylaminopurine and 2.0 mgl^-1 naphthaleneacetic acid. Regal geranium cultivars Tiny Tot and Lavender Grand Slam were shown to produce between 2–50 shoot primordia per explant when initiated on the same medium.     

In vitro zygotic embryo culture of an endangered forest tree Givotia rottleriformis and factors affecting its germination and seedling growth

Summary This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 gl^−1 sucrose. A sucrose concentration lower or higher than 30 gl^−1 resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mgl^−1 tyrosine in the medium. Seedlings germinated in the presence of 0.2–0.4 mgl^−1 α-naphthaleneacetic acid and 0.3–0.5 mgl^−1 indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60–70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.     

Micropropagation of sugarcane (Saccharum spp.)

A method for callus induction, adventitious bud regeneration, shoot multiplication and rooting of in vitro formed shoots of Helianthus annuus L. var. Argentario is described. Hypocotyl and cotyledon explants formed callus on medium containing 2 mgl^–1 naphthalene acetic acid and 0.5 mgl^–1 benzyladenine. Adventitious buds were formed on hypocotyl segments on medium containing 0.5–2 mgl^–1 benzyladenine. The optimal level of sucrose concentration for shoot regeneration from hypocotyls was 1.5%. Multiplication from shoot apices was promoted by kinetin (2 mgl^–1) plus gibberellic acid (5 mgl^–1), benzyladenine (2 mgl^–1) plus gibberellic acid (10 mgl^–1) or at lower frequency by benzyladenine (1 mgl^–1). A general feature of the plantlets formed in vitro was the precocious flowering.     

Induction of mitosis in polytene nuclei and hormonal effect on nuclear changes during callus initiation in diploid Urginea indica Kunth. (liliaceae)

Bulb scale and inflorescence explants of Urginea indica Kunth. (2n=20) were cultured in vitro on modified Murashige & Skoog's medium with different hormonal composition. Media containing 2,4-dichlorophenoxy-acetic acid (2,4-D) (2 and 4 mgl^–1) and -naphthalene-acetic acid (NAA) (2 mgl^–1) could induce callus in inflorescence explants. Combination of 2,4-D (4 mgl^–1) + NAA (2 mgl^–1) + Kinetin (2 mgl^–1) only could induce callus formation in scale explants. The bulb scale explants contained mostly diploid cells while the inflorescence explants contained cells with nuclear DNA content ranging from 2C to 64C. The lowest karyological heterogeneity was recorded in callus derived from bulb scale and in callus derived from inflorescence induced with NAA. The highest variability was recorded on media with 2,4-D alone. Induction of division, probably of the pre-existing polytenic nuclei in the inflorescence explant, has been suggested to be the cause of origin of polyploid cells in such cases.     

An efficient plant regeneration system through callus for Pseudarthria viscida (L.) Wright and Arn., a rare ethnomedicinal herb

The purpose of this study was to develop a protocol to induce high frequency of callus and subsequent plantlet regeneration for Pseudarthria viscida; an important medicinal plant. The cotyledonary node and young leaf pieces (1 × 0.5 cm, length × breadth) were used as explants for callus induction and subsequent shoot regeneration and adventitious roots induction from the shoots. The best results were achieved on the following media: (1) 96 % callus induction from cotyledonary node explants on MS medium supplemented with 1.5 mgl⁻¹ 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgl⁻¹ 1-naphthalene acetic acid (NAA), (2) 97 % shoot regeneration from cotyledonary node derived calli with an average of 44.9 shoots per explant on MS medium fortified with 3.0 mgl⁻¹ N₆-benzylaminopurine (BA) and 1 mgl⁻¹ NAA,37 (3) 98 % rooting with an average number of 3.3 roots per shoot on MS medium containing indole-3-butyric acid (IBA) or NAA (0.5–4 mgl⁻¹) after 45 days. The plantlets were transferred to the field after acclimatization. Of the 40 plantlets transplanted to the soil, 29 survived (72.5 %).     

Effectiveness of SC2053 as a chemical hybridizing agent for winter wheat

The use of chemical hybridizing agents (CHA) allows production of hybrid wheat seeds. We evaluated the effectiveness of a new CHA (SC2053) to induce male sterility on winter wheat in controlled growth conditions. CHA effectiveness was measured with the application of 4 doses (0–400–700–1000 g.ha^–1) at 7 stages. These stages were defined by the length of the main stem spike (1–4–7–11–15–20–40 mm). At heading, individual ears were isolated with a greaseproof paper bag. The seeds formed were counted on treated and control ears. The spikes' sterility was calculated three weeks after flowering. The sterility of the main stem's spike reached 95% to 100% for application of 700 g.ha^–1 and 1000 g.ha^–1 for main stem spike length of 7 mm to 20 mm. The effects of ear tillering (5 tillers per plant) on CHA effectiveness were also investigated. We observed a significant delay of ear development between the main stem and tillers so that complete sterilities were not reached for each dose. Since tillering in field conditions rarely exceeds 3 ears per plant, CHA effectiveness was studied on plants bearing 3 ears. The mean sterility of the first 3 ears was close to 100% for applications with 700 g.ha^–1 and 1000 g.ha^–1 at stages from 11 mm to 20 mm of main stem spike length.     

Secondary somatic embryogenesis and plant regeneration in cassava

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somatic embryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 24–30 days on Murashige and Skoog basal medium supplemented with 2–8 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (Stage I medium) before transfer to medium supplemented with 0.01 mgl^-1 2,4-D and 0.1 mgl^-1 6-benzylamino purine (BAP) (Stage II medium). Under these conditions, secondary somatic embryos developed directly from the cotyledons and shoot-tip region of primary somatic embryos by a developmental process morphologically very similar to that occurring on zygotic cotyledon explants. Apical shoot extension and adventitious root formation occurred when somatic embryos were isolated from parental cultures and incubated on Stage II medium. Somatic embryo-derived plants growing in greenhouse conditions appeared morphologically normal when compared with non-regenerated plants.     

Direct somatic embryogenesis from protoplasts of Citrus mitis Blanco

Protoplasts isolated from embryogenic suspension cultures of Citrus mitis were cultured in a medium without any plant growth substances. Somatic embryos developed directly from protoplasts without an obvious intervening callus phase. As many as 1,800 somatic embryos developed from 4 ml of protoplast suspension (density 2×10⁶/ml) cultured for 35 days. Upon transferring the embryoids to medium with 1 mgl^–1 GA₃, they developed into plant-lets. Rooted plantlets were obtained in 3 months after protoplast isolation.Abbreviations BAP Benzylaminopurine - GA₃ Gibberellic acid - MT Murashige and Tucker medium (1969) - FDA Fluorescein diacetate     

The importance of initial seed size in wheat plant response to salinity

Large initial seed size frequently confers distinct advantages on cereal crops in terms of seedling vigor, hardiness, improved stand establishment, and higher productivity. This study was conducted to determine if these advantages inherent in the plants grown from large seeds persist when the crop is subjected to salinity stress. Two hard red spring wheat cultivars, Yecora Rojo and Anza were grown in greenhouse sand cultures from seed of two size classes that differed in weight by a factor of 2. The cultures were irrigated four times daily with complete nutrient solutions to which NaCl and CaCl₂ (2:1 molar ratio) were added to achieve osmotic potentials of –0.05. –0.55, and –0.70 MPa with electrical conductivities of 1.8, 12.8, and 15.8 dS m^-1, respectively. In response to both salinity and small initial seed size, the following plant characteristics decreased: leaf appearance rate, blade area, tillers per plant, spikelets per spike and seeds per spike. Plants grown from large seeds out-yielded those from small seeds by 8, 37, and 27% for Yecora Rojo and by 15, 30, and 23% for Anza at osmotic potentials of –0.05, –0.55 and –0.70 MPa, respectively. Compared to the corresponding nonsaline controls, the yield of Yecora Rojo grown at –0.55 MPa was 51% for the plants from large seed and 35% from the small seeds. For Anza salinized at –0.55 MPa, these values were 49 and 40%, respectively. Exploitation of the benefits derived from large initial seed size may be a cost-effective management strategy for improving wheat productivity in salt-affected areas.     

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of coriander (Coriandrum sativum L.)

Hypocotyl segments and zygotic embryos of coriander formed embryogenic calli at frequencies of up to 75% when cultured on MS medium supplemented with 1 mgl^–1 2,4-D. Calli were transferred to MS liquid medium with 1 mgl^–1 2,4-D to initiate cell suspension cultures. Embryogenic cells became finely dispersible in the medium as the subculture proceeded. Cultures were transferred to a nitrogen compound enriched liquid MS medium containing 2% sucrose and 0.1 mgl^–1 2,4-D, and cultured two weeks before plating on MS basal medium. Approximately 75% of cell aggregates (1 to two mm in diameter) underwent development into globular to cotyledonary somatic embryos after two weeks of plating. Most of the embryos were subsequently regenerated into plantlets. Regenerants were successfully transplanted to potting soil and grown to maturity in a phytotron.Abbreviations MS Murashige and Skoog - MS1D MS medium + 1 mgl^–1 2,4-D     

Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), 'MPB-Bobwhite 26', and an elite durum wheat (Triticum turgidum var. durum L.), 'Mexicali', were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5 mg l^–1 2,4-D for 45 days produced callus and plant regeneration in 'MPB-Bobwhite 26', but lower callus yield and plant regeneration in 'Mexicali', indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in 'Mexicali' embryos that were incubated in media containing 2 mg l^–1 2,4-D and 2 mg l^–1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use of NaCl in the callus formation could form the basis for improved transformation of durum wheat varieties.     

In vitro plantlet formation from juice vesicle callus of satsuma (Citrus unshiu Marc.)

Callus was induced from juice vesicles of satsuma mandarin on Murashige & Skoog medium supplemented with -naphthaleneacetic acid (NAA), kinetin (K) and gibberellin (GA). Adventitious embryoids arose from the callus tissue on the medium containing 1 mgl^–1 NAA alone. The embryoids grew into embryos which resulted in a plantlet on medium containing 1 mgl^–1 GA.Abbreviations GA gibberellin - K kinetin - NAA -naphthaleneacetic acid     

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.     

Somaclonal variation in some agronomic and quality characters in wheat

Summary A total of 256 selected lines derived from tissue culture of three hexaploid wheat cultivars were grown in a replicated hill plot experiment to examine somaclonal variation in agronomic characters. The lines were derived by single plant selection for various characters from a total of 100 regenerants, and were either SC₃ or SC₄ generation in the test. Significant variation was found in all the characters measured: height, grain number per spike, kernel weight, yield, total dry weight and harvest index. In most cases, variation could be identified which was both less than and greater than the parental controls. However, there was also an apparent effect of the parent cultivar on the total amount and direction of the variation. For two cultivars, lines could be traced back through the culture phase to individual explant embryos. Many of the original embryos contributed significant variation, and most characters showed significant variation arising from more than one embryo. In the following year, 32 lines selected from the hill plot experiment were grown in larger replicated plots and yield, harvest index and a number of grain and baking quality characters were measured. No lines selected for high yield or harvest index maintained significant improvements over their parental controls. However, significant variation was displayed for many of the quality characters examined. Significant increases in kernel weight, hardness and protein content, and a significant reduction in yellow pigmentation represented potentially useful improvements. Only unfavourable variation was seen in flour yield and in mixograph height, time and breakdown.     

In vitro regeneration of Camellia sinensis (L.) O. Kuntze by somatic embryogenesis

Within 3 weeks of culture, excised cotyledon expiants of Camellia sinensis (L.) O. Kuntze produced somatic embryos without intermediate callus when cultured in Murashige and Skoog's basal medium with 30 g^–1 sucrose. In medium without plant growth regulators, up to 60% of the cultures developed somatic embryos. Embryogenic competence was reduced by increasing concentrations of plant growth regulators tested (i.e. kinetin, 6-benzylaminopurine, and indole butyric acid). The somatic embryos developed, grew to maturity without being subcultured within 6–8 weeks. Secondary embryogenesis was not observed. Germination of isolated mature somatic embryos was low in medium without plant growth regulators. Up to 53% and 60% germination occurred when medium impregnated with kinetin at 1.8 mgl^–1 or 1.0 mgl^–1 6-benzylaminopurine were used respectively. Callus was also routinely produced when cotyledons were cultured in MS basal medium with auxins (2,4-dichlorophenoxyacetic acid and indole acetic acid). Callus induction was however, also achieved in plant growth regulator free medium. Indirect somatic embryogenesis was not induced in the present study.Abbreviations K kinetin - BAP 6-benzylaminopurine - IBA indole butyric acid - IAA indole acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - Fe-EDTA Ethylenediaminetetra-acetic acid (Ferric monosodium salt)     

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium