Adventitious shoot regeneration from cultured petal explants of carnation

Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 M BA with or without 5 M NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N-phenylurea and N-1,2,3-thiadiazol-5-yl-N-N-phenylurea were more effective than BA, kinetin, N⁶-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - GA₃ gibberellic acid - 2iP N⁶-2-isopentenyl adenine - KT-30 N-2-chloro-4-pyridyl-N-phenylurea (also called 4PU) - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. Hale's Best Jumbo in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 M indole-3-acetic acid and 5 M benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 mol m^-2 s^-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.     

Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

Conditions for plant regeneration from excised cotyledons of Vigna radiata were studied. Complete plant developed from the uncallused proximal ends of cotyledons on Murashige & Skoog's (MS), Gamborg's (B₅) and C (MS salts + B₅ vitamins) basal media. The basal medium C was found to be best for plant regeneration. Regeneration frequency, however, varied with genotype, size, orientation and age of explant and the different plant growth regulators combination in the medium. Addition of cytokinins induced callusing at the proximal ends of cotyledons followed by multiple shoot formation. Out of 6-benzyl aminopurine (BAP), kinetin (KIN), N (^–2 isopentyl) adenine (2iP) and adenine sulphate (AS), only BAP and KIN were found to be more effective in enhancing the frequency of shoot regeneration. BAP at 1×10^-1M induced maximum (60%) shoot regeneration whereas maximum number of shoots (8 to 9 shoots) per explant was observed with 5×10^-6M BAP. Cotyledons excised from two-day old seedlings were most regenerative. The regenerative response of cotyledons decreased when sliced into two equal parts either longitudinally or transversely. Callusing and organogenic differentiation occured only if the petiolar end of cotyledons was in contact with medium. None of the tested treatments were effective in inducing shoot bud differentiation from subcultured callus. Well developed shoots rooted when incubated on half strength MS, MS and MS basal medium supplemented with IAA (5×10^-6M). The rooted plants were transferred to pots and later established in the field with 60% success.Abbreviations AS adenine sulphate - BAP 6-benzylaminopurine - B₅ medium after Gamborg et al. [6], - C Medium with MS salts + B₅ vitamins - 2iP N (^–2 isopentyl) adenine - IAA indole-3-acetic acid - KIN Kinetin - MS medium after Murashige & Skoog [21] - NAA 1-napthaleneacetic acid     

Adventitious shoot formation from leaf explants of Rhododendron

A protocol for high frequency adventitious shoot regeneration adventitious shoot regeneration from leaf explants of Rhododendron spp. has been developed. The highest percentage of regeneration and the greatest number of shoots were obtained when leaf explants were cultured on Anderson's medium containing 4.9 M IBA and 73.8 M 2iP. Genotypic variation was observed for adventitious shoot regeneration potential among the seven cultivars tested. Regeneration frequencies ranged from 0 to 96%. Lodestar had the highest rate of regeneration after 3 months of culture with 96% shoot regeneration and an average of 14 shoots per explant. Regenerated shoots were rooted in soil in about 2 months. This protocol should be useful in applying gene transfer techniques to Rhododendron improvement.Abbreviations IAA 1-H-indole-3-acetic acid - NAA 1-naphthaleneacetic acid - IBA 1-H-indole-3-butyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - 2iP N-(3-methyl-2-butenyl)-1-H-purine-6-amine     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

A simple protocol for micropropagating diploid and tetraploid watermelon using shoot-tip explants

Shoot-tip explants from 21-day-old aseptically-germinated watermelon seedlings were incubated on solidified MS medium containing test concentrations of benzyladenine (BA) and kinetin (each at 0, 1, 5 or 10 µM), and thidiazuron (TDZ; 0, 0.1, 1 or 5 µM) for 8 weeks. Approximately 1.5x–2.8x more axillary shoots formed at the optimum BA level (1 µM) compared to the best TDZ (0.1 µM) or kinetin (10 µM) concentration. The ability of various diploid and tetraploid genotypes to undergo prolonged axillary shoot proliferation on medium with 1 µM BA was examined. Among the genotypes tested, the number of axillary shoots per explant was greater for Bush Jubilee and Jubilee II than for Minilee, Dixielee, and the tetraploid genotypes. For a majority of the genotypes tested, the number of shoots per explant was low (2.7–4.0) during the first month of culture, peaked (5.3–12.5) at 2 to 3 months, and then declined (3.7–7.7) at 6 months. In contrast, the number of shoots per explant was greatest (11.7) for Bush Jubilee during the first month of culture and declined to 7.7 by the sixth subculture. The percentage of rooted shoots varied from 60% to 100% and the percentage of acclimatized plants ranged from 21% to 96% depending on the genotype and the length of time in culture. Using this procedure, 13,200 finished plants could be produced in 3 months from 250 seedlings.This process is protected by United States patent No. 5,007,198. Those interested in using this technology must secure a license from the University of Florida.     

Uptake and metabolism of benzyladenine during shoot organogenesis in Petunia leaf explants

Benzyladenine (BAP) uptake and metabolism were characterized during the key stages of shoot organogenesis in leaf explants of Petunia MD1. Using leaf explant transfer experiments, it was shown that exposure to 2.2 M BAP for 6, 8 or 10 days induced shoot formation on 27, 80 and 100% of the explants respectively, with a concomitant increase in the number of shoots per explant. BAP uptake and metabolism were characterized in leaf explants after 1, 3, 6 or 10 days exposure to [³H]BAP or 10 days exposure plus an additional 2 days on basal medium (10+2). BAP and 9--D-ribofuranosyl-BAP ([9R]BAP) were detected at days 1 and 3 only. Therefore, the BAP free base was not detectable during the shoot induction period between days 6 and 10, as defined by leaf transfer experiments. The BAP ribotide pool was largest on day 1 and decreased to day 10+2. It is possible that the BAP ribotide pool provided either the active cytokinin itself or acted as a short-term storage form for the active cytokinin in petunia shoot organogenesis. Other metabolites detected in petunia leaf tissue included 7--D-glucopyranosyl-BAP ([7G]BAP), 9--D-glucopyranosyl-BAP ([9G]BAP) and an unidentified metabolite C.Abbreviations BAP benzyladenine - [7G]BAP 7--D-glucopyranosyl-BAP - [9G]BAP 9--D-glucopyranosyl-BAP - [9R]BAP 9--D-ribofuranosyl-BAP - [9R-5P]BAP 5-monophosphate of [9R]BAP - [9R-5PP]BAP 5-diphosphate of [9R]BAP - [9R-5PPP]BAP 5-triphosphate of [9R]BAP - TEA Triethylamine This research was supported in part by NSF Grant DCB-8917378 to J.D.C. and USDA-CRGO Grant 89-37261-4791 to T.J.C.     

In vitro regeneration of evergreen azalea from leaves

Rhododendron simsii Hellmut Vogel was regenerated using different types of explants, auxins and cytokinins. After a callus induction phase, with 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid, adventitious shoot regeneration was obtained on a medium supplemented with thidiazuron or zeatin. With thidiazuron shoots were small and a subsequent elongation step was required before rooting. An elongation step was not required when zeatin was used. The duration of the callus induction phase was negatively correlated with the regeneration capacity.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - 2iP 6-(--dimethylallylamino)purine - NOA ß-naphthoxyacetic acid - BA 6-benzyladenine - IBA 1-H-indole-3-butyric acid - IAA 1-H-indole-3-acetic acid - TDZ thidiazuron - WPM woody plant medium     

Glycerol-, inositol-, and reducing end hexose-containing oligosaccharides in human urine

Ten previously unreported oligosaccharides have been purified from the urines of human subjects using a combination of gel filtration, ion exchange, and thin-layer chromatographies. Their structures were determined by direct probe mass spectrometry, methylation analysis, and proton NMR spectroscopy of the permethylated oligosaccharide alditols.On the basis of composition, the oligosaccharides could be divided into three groups. Five oligosaccharides containing glycerol were characterized as glucosyl1-1glycerol; glucosyl1-1glycerol; galactosyl1-1glycerol; glucosyl-1-1(fucosyl-1-2)glycerol and/or fucosyl-1-1(glucosyl-1-2)glycerol; and glucosyl-1-1(galactosyl-1-2)glycerol or galactosyl-1-1(glucosyl-1-2)glycerol. Four inositol-containing oligosaccharides were characterized as galactosyl1 (fucosyl1)inositol,N-acetylgalactosaminyl1 (fucosyl1)inositol, fucosyl1-2galactosyl1 (N-acetylgalactosaminyl1)inositol and fucosyl1-2galactosyl1-4-N-acetylglucosaminyl1(N-acetylgalactosaminyl1)inositol. Finally, galactosyl1-3(fucosyl1-2)galactosyl1-6galactosyl1-4(fucosyl1-3)glucose, an oligosaccharide with glucose at its reducing end, was tentatively identified. The significance and possible origins of the carbohydrate structures are discussed.     

Genotypic differences in cold tolerance are masked by high sucrose and cytokinin in shoot cultures of sugarbeet

Cold tolerance of field grown plants and shoot cultures of a commercial sugarbeet cultivar, Hilma, was compared with that of two cultivars bred for improved cold tolerance, Monofeb and Winter Hybrid 88619. Leaves of Monofeb and Winter Hybrid 88619 showed an increase in frost tolerance compared to Hilma, as assessed by electrolyte leakage measurements, in both July, and November. However, all varieties exhibited acclimation in the latter month. Similar qualitative differences between cultivars were detected in shoot cultures only when maintained on low (1%) sucrose medium, without added plant growth regulators. The use of high (3%) sucrose and benzyladenine, which releases apical dominance producing multiple shoots, each contributed to a substantial lowering of the temperature at which cold-induced damage occurred in leaves. Under these conditions varietal differences were masked. The implications of these findings in regard to in vitro selection for improved cold tolerance in organized cultures are discussed.Abbreviations BA benzyladenine - MS Murashige & Skoog (1962)     

Identification of the cytokinins in red pine seedlings

Zeatin-9-riboside was identified in shoots and roots of Pinus resinosa by GC-MS analysis of its permethyl derivative. Based on their chromatographic properties on Sephadex LH-20 and C₁₈ HPLC, and their susceptibility to enzymatic degradation, several other cytokinins have been tentatively identified. The basic fraction of both the roots and shoots contained zeatin, whereas the shoots contained dihydrozeatin-O-glucoside and the roots contained zeatin-O-glucoside. Zeatin-9-riboside monophosphate, isopentenyladenosine monophosphate ([9R-5P]iP) and glucosyl phosphate derivatives were detected in the acidic fractions from both roots and shoots. There were equivalent amounts of [9R-5P]iP in both roots and shoots. The presence of equivalent amounts of [9R-5P]iP in both the roots and shots suggests that cytokinin biosynthesis may be occurring in both locations.Abbreviations AMP adenosine-5-monophosphate - BAP benzylaminopurine - BSA bovine serum albumin - BuOH butan-1-ol - CK cytokinin - (diH)Z dihydrozeatin - (diH OG)Z dihydrozeatin-O-glucoside - (diH OG)[9R]Z dihydrozeatin-9-riboside-O-glucoside - DW dry weight - EtOH ethanol - FW fresh weight - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - [9R]iP isopentenyladenosine - [9R-5P]iP isopentenyladenosine monophosphate - MeOH methanol - PVP polyvinylpyrrolidone - RFE rotary film evaporation - TEAB triethyl ammonium bicarbonate - Z zeatin - [9R]Z zeatin-9-riboside - (OG)Z zeatin-O-glucoside - [7G]Z zeatin-7-glucoside - [9R-5P]Z zeatin-9-riboside monophosphate     

Genetic analysis of brown planthopper resistance in twenty varieties of rice,Oryza saliva L.

Summary The inheritance of resistance to brown planthopper, Nilaparvata lugens (Stol.), of 20 rice cultivars was studied. Single dominant genes that are allelic to Bph 3 condition the resistance in cultivars Ptb 19, Gangala (Acc. 7733), Gangala (Acc. 15207), Horana Mawee, Kuruhondarwala, Mudu Kiriyal and Muthumanikam. Single recessive genes that are allelic to bph 4 govern the resistance in cultivars Gambada Samba, Heenhoranamawee, Hotel Samba, Kahata Samba, Kalukuruwee, Lekam Samba, Senawee, Sulai, Thirissa and Vellai Illankali. The resistance in Ptb 33, Sudu Hondarwala, and Sinna Sivappu is governed by one dominant and one recessive gene which segregate independently of each other. The dominant resistance genes in these cultivars appear allelic to either Bph 1 or Bph 3. Similarly, the recessive genes in these cultivars seem allelic to either bph 2 or bph 4. Further investigations are needed to conclusively determine the allelic relationships of resistance genes in Ptb 33, Sudu Hondarwala and Sinna Sivappu.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Organogenesis from ‘Passe Crassane’ and ‘Old Home’ pear (Pyrus communis L.) protoplasts and isoenzymatic trueness-to-type of the regenerated plants

Summary Large numbers of highly viable mesophyll protoplasts were isolated from shoot cultures of the scion cv Passe Crassane and the rootstock genotype Old Home of common pear (Pyrus communis L.). Protoplasts were cultured for both genotypes either as liquid layers or as liquid-over-agar cultures, in ammonium-free MS medium with 0.5 M mannitol, 50 mg/l casein enzymatic hydrolysate (CEH), 2.0 mg/l NAA and 1.0 mg/l BAP, plus either 0.5 mg/l IAA (for Old Home) or 2.0 mg/l IAA (for Passe Crassane). Protoplast microcalli, obtained by day 60 (Passe Crassane) or day 80 (Old Home), were transferred for further growth to ammonium-free MS medium with 2.0 mg/l NAA and 1.0 mg/l BAP. Shoot bud regeneration from the protoplastderived callus was first attempted between 100 (Passe Crassane) and 120 (Old Home) days after protoplast isolation. For Passe Crassane, shoot buds were regenerated (day 130) on a half-strength MS medium with 0.1 mg/l IBA, 0.5 mg/l BAP, 50 mg/l CEH and 20 mg/l Ca-panthotenate. For Old Home, shoot but regeneration only occurred 30 days later and on the same medium as above, which was additionally supplemented with double the concentration of the group B vitamins found in the original MS formulation and 0.05 mg/l GA₃. Following micropropagation and in vitro rooting of shoots, the plants were transferred to soil following standard procedures. Trueness-to-type of the regenerated plants was assessed by analysing their leaf isozyme banding profiles (for EST, AP, PRX, SOD, ENP, LAP, PGI, AAT, ADH, MDH and PGM) and comparing them to those corresponding to the original shoots that provided the protoplasts. No differences between the mother shoots and the protoclones were observed for any one of the 11 isozyme systems studied.     

Somatic embryogenesis,plant regeneration and somaclonal variation in barley

In vitro culture of immature embryo and young leaf tissues was carried out with five cultivars of barley, Hordeum vulgare. Two cultivars (Albacete and Porthos) responded poorly from both types of explants, while the three others (Dissa, Golden Promise and Ingrid) produced a high frequency of embryogenic callus from these explants (25–60%). For Dissa and Ingrid, young leaf explants were slightly better than immature embryo explants for embryogenic callus induction, while immature embryo cultures of Golden Promise responded better than young leaf explants. Thus, there appears to be a significant genotype × explant interaction in the initiation of embryogenic callus in barley.Some phenotypic variants were detected among the regenerated plants of Golden Promise and Ingrid, most originating by epigenetic changes. Only in one case was the variant phenotype heritable, probably due to a mutation in the chloroplast DNA. Mitotic alteractions were not detected. Consequently, somaclonal variation did not appear to be a very frequent event in plants regenerated from 1- to 6- month-old cultures of barley.     

The effect of leaf source and developmental stage on shoot organogenic potential of sweetgum (Liquidambar styraciflua L.) leaf explants

Leaf explants harvested from shoot proliferating cultures and intact plants of Liquidambar styraciflua Variegata were placed on solidified Woody Plant medium supplemented with 0.1 mgl^-1 (0.5 M) naphthaleneacetic acid and 2.5 mgl^-1 (11.1 M) benzyladenine to initiate shoot meristems directly. Leaves from intact plants produced over 4 times more adventitious shoots than leaves from in vitro shoots and had a greater tendency to form shoots on the lamina. The relative developmental age of leaf tissue used dramatically influenced the shoot organogenic response observed for leaf explants from intact plants of L. styraciflua Variegata and Moraine.-Leaves that were either 20% or 50% of full size and still actively expanding were superior to other developmental stages for shoot organogenesis. As developmental leaf age increased throughout the period of leaf expansion, the number of shoots forming on the petiole stub remained constant, whereas shoot formation on the lamina increased 8 fold. Shoots derived from Variegata leaves rooted well and grew normally as plants. Differences in rooting ability and plant size could be detected between groups that had been separated according to explant source (in vitro vs. intact plant) and the location of shoot formation (petiole vs. lamina).     

Plant regeneration and genetic transformation of Lotus angustissimus

Culture conditions have been established for callus induction and growth from different explants in L. angustissimus L. Calli were obtained from hypocotyls, leaves, stems, cotyledons and roots cultured on media containing 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid with kinetin, N⁶ – ² or benzyladenine in different combinations and concentrations. Only those calli induced in presence of -naphthaleneacetic acid with benzyladenine or kinetin produced shoots. Calli induced from hypocotyl explants were the most efficient in regeneration of shoots. Transformation with an Agrobacterium rhizogenes binary vector carrying the plasmid pBI 121.1 is reported. The percentage of cotransformation was estimated by testing GUS activity in hairy roots. The integration of Ri T-DNA and the NPTII gene in transformed plants was confirmed by molecular analyses and in vitro culture of transgenic tissues in the presence of kanamycin.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - 1AA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2iP N⁶ – ² - PA proanthocyanidins - NOS nopaline synthase - NI TII neomycin phosphotransferase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

Shoot,root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 M concentrations. Direct shoot formation occurred on media with 10 M kinetin and 0.001 M IAA or NAA. Root formation was observed on media with 0.1–10 M IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 M IAA and 0.1 M kinetin. Shoot organogenesis was increased by substituting 10 M zeatin or N⁶-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (Red Alert) to 5.3 (Large Red Cherry). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.Abbreviations BA N⁶-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2iP N⁶-[²-isopentyl]adenine - NAA -naphthaleneacetic acid - PGR plant growth regulator