Use of lysozyme for treatment of bacterial contamination in <Emphasis Type="Italic">in vitro</Emphasis> shoot cultures of fruit plants

Summary Use of lysozyme was tested for treatment of bacterial contaminations in in vitro shoot cultures of quince (Cydonia oblonga) ‘BA 29’ and the hybrid (Prunus persica × P. amygdalus) rootstock ‘GF 677’. Shoots which had been contaminated for about 1 yr by Bacillus circulans and Sphingomonas paucimobilis were treated in liquid culture, at pH 4.5, with 9–36 mg ml⁻¹ egg white lysozyme (EWL), and compared to each other and to untreated cultures for their growth, proliferation, and number of bacterial colony-forming units in the tissues. EWL did not negatively affect shoot growth up to 18 mg ml⁻¹; furthermore, the proliferation rates of EWL-treated shoots were sometimes higher than those of controls. In contrast, the concentration of 36 mg ml⁻¹ had some deleterious effect on the regrowth capacity and shoot production of ‘GF 677’ at the first subculture to solid medium after EWL, treatments. EWL had a simple bacteriostatic effect against Sphingomonas paucimobilis; in contrast, it was effective at 18 mg ml⁻¹ in eliminating Bacillus circulans in both ‘BA 29’ and ‘GF 677’ cultures, after optimal treatment duration.     

Acidogenic growth model of embryogenic cell suspension culture and qualitative mass production of somatic embryos from triploid bananas

A young male flower-derived embryogenic suspension cell population of AAA ‘Pei Chiao’, ‘Dwarf Cavendish’, and AAB ‘Raja’ was used for developing an acidogenic growth model . We hypothesized that a close relationship exists between the self-regulated pH medium and the corresponding changes in the growth phases. Studies have reported that a pH below 4.6 may prevent the embryogenic cells from undergoing polar growth. Controlling pH up to a level 4.6 within 2 days during the changes of pre-embryogenic cells (PECs) and proembryogenic masses into embryogenic determined cells (EDCs) uniformly resulted in unequal cell division. The hydrogen ion buffer 2-N-morpholino-ethanesulfonic acid at 10 g L⁻¹ was added to MA2 and MA3 media, showing the medium pH of MA3 up to 5.0, thus maintaining a relatively stable pH in AAA ‘Pei-Chiao’ and AAB ‘Raja’ cells that autoregulate acidification, significantly increasing the number of somatic embryos. When the proliferation and globularization phases were acidified to pH 3.5 ± 0.2, cells were released to free single cells of PECs and EDCs after 21 days. This study provides possible explanation that PECs deposit callose on their cell walls as a possible protector from strong acidic condition. Regulation at pH 5.0 ± 0.2 resulted the production efficiency achieved was 0.9 million somatic embryos per 1 mL of the settled cell volume.

     

Cleansing the long-term micropropagated triploid watermelon cultures from covert bacteria and field testing the plants for clonal fidelity and fertility during the 7–10 year period in vitro

The study was aimed primarily at cleansing the in vitro-decline displaying long-term micropropagated triploid watermelon ‘Arka Manik’ cultures from covert bacteria and their further field testing. Disinfectant treated shoots showed endophytic survival but to a lesser extent in shoot-tips. Culturing the NaOCl (5 min) treated shoot-tips on filter paper bridges in liquid watermelon medium containing single antibiotic (gentamycin (Gm), amoxycillin (Ax) or cefazolin (Cz) at 50, 250, 500 or 1000 mg l⁻¹) for 1 month followed by repeated indexing of medium and tissue for two-four subculture passages facilitated the cleansing of cultures with 12.5% recovery as monitored for 2 years. Partial bleaching damage by NaOCl, phytotoxicity to higher levels of antibiotics, poor growth response in the initial sucrose-free medium and rampant hyperhydricity came in the way of a higher recovery. The effectiveness of the above approach was ascertained after back inoculating clean cultures with a mixture of Gram-positive and Gram-negative bacteria yielding 30, 45 and 35% clean cultures in Gm, Ax and Cz (250 mg l⁻¹ for 1 month) treatments, respectively in modified medium compared with 10% recovery after mere HgCl₂ surface sterilization. The results indicated that antibiotic treatment was essential but not its choice, and extended culture-indexing subsequent to disinfection or antibiotic treatment was crucial in identifying clean stocks. Cleansed cultures, showed restoration of growth but a drop in rooting. Most of the in vitro cultures appeared normal and true-to-type during the 7–10 year period in vitro but a small proportion of bacteria-harboring stocks displayed ‘epigenetic variations’. Acclimatized plants and those in the field also appeared true-to-type but for a minor proportion derived from bacteria-harboring stocks. Field-plants which originated from bacteria-freed stocks after 9 years of continuous culturing were normal and fertile validating the possibility of keeping treasured cultures in vitro for long periods if covert contaminants are checked.     

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.     

Chemical and physical-chemical characteristics of dietary fibres from Ulva lactuca (L.) Thuret and Enteromorpha compressa (L.) Grev.

Dietary fibres from Ulva lactuca (L.) Thuret (sea lettuce) and Enteromorpha compressa (L.) Grev. (A.O. nori) were measured according to a ‘standard’ method and a ‘physiological’ protocol simulating the gastric and intestinal environments. U. lactuca contained 15.8–8.0% soluble and 24.2–32.6% insoluble fibres according to the ‘standard’ and ‘physiological’ methods, respectively. For E. compressa, these values were 14.9–15.9 and 21.6–28.7%, respectively. For both algae, the composition suggests that the soluble fibres were xylorhamnoglycuronans sulphates and insoluble fibres were essentially composed of glucans. No marked chemical compositional variation was observed between soluble fractions extracted under the simulated gastric and intestinal conditions. Fibres in both algae are hydrophilic but the water holding capacities were higher after extraction of soluble fibres (5.5–9.5 g g⁻¹ for the dry algae; 14.0–16.0 g g⁻¹ for the standard insoluble fibres). Water soluble fibres demonstrated low intrinsic viscosities at 37 °C in buffers, particularly those from E. compressa (36.0–36.5 ml g⁻¹), and was affected by pH for those of U. lactuca (147.5 ml g⁻¹ at pH 3.0 and 175.0 ml g⁻¹ at pH 7.3).     

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.     

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.     

Production of imidazole alkaloids in cell cultures of jaborandi as affected by the medium pH

The effect of pH (from 4.8 to 9.8) on the production of pilosine and pilocarpine and on their partition between cell and medium was studied in two lineages (P and PP) of Pilocarpus microphyllus cell suspension cultures. Highest mass accumulation was observed at high pHs and both lineages produced pilocarpine while only lineage PP produced pilosine. Both alkaloids were released in the medium but higher accumulation occurred in the cells. The highest production of pilocarpine was at pH 8.8–9.8 in both cell lineages. Other imidazole alkaloids were also identified in both lineages. At all pHs tested, the pH in the media cultures tended to stabilize around 6 after 10–15 days of cultivation. NO₃ ⁻ and NH₄ ⁺ variation in the media might partially explain the pH stabilization.     

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.     

Strawberry sepal: Another explant for thidiazuron-induced adventitious shoot regeneration

Summary An efficient system to regenerate shoots on excised sepals (calyx) of greenhouse-grown ‘Bounty’ strawberry (Fragaria x ananassa Duch.) was developed in vitro. Sepal cultures produced multiple buds and shoots without an intermediary callus phase on 2–4 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ)-containing shoot induction medium within 4–5 wk of culture initiation. Young expanding sepals with the adaxial side touching the culture medium and maintained for 14 d in darkness produced the best results. In a second experiment, sepals proved more effective than the leaf discs and petiole segments for regenerating shoots. A third experiment compared the effects of six concentrations of two cytokinins (TDZ at 0, 0.5, 2, and 4 μM and zeatin at 2 and 4 μM) for elongation of sepal-derived adventitious shoots. The media containing TDZ generally promoted more callus formation and suppressed shoot elongation. TDZ-initiated cultures transferred into the medium containing 2–4 μM zeatin, produced usable shoots after one additional subculture. Shoots were rooted in vitro in the same medium used for shoot regeneration, but without any growth regulators. When transferred to potting medium, 85–90% of in vitro plantlets survived.     

Medium,container and genotype all influence in vitro cold storage of apple germplasm

The goal of this study was to evaluate the in vitro storage of apple germplasm by screening a range of genotypes followed by more comprehensive testing of multiple parameters on two genotypes of differing species, Malus domestica cultivar Grushovka Vernenskaya and wild Malus sieversii selection TM-6. Stored plants were rated on a 6 point scale (0 low to 5 high) for plant appearance at 3 month intervals after storage at 4°C. Combinations of carbon source (sucrose and/or mannitol), nitrate nitrogen content (25, 50 or 100%) and plant growth regulators (ABA, BAP, IBA) were studied in three types of containers (tissue culture bags, test tubes or jars). An initial screen of 16 genotypes stored in tissue culture bags indicated that plantlets could be stored at 4°C for 9–14 months without subculture on standard 3% sucrose Murashige and Skoog (1962) (MS) medium with no plant growth regulators (PGRs). In subsequent in-depth studies on the two genotypes, ANOVA indicated highly significant interactions of medium, container and genotype. ‘Grushovka Vernenskaya’ shoots with no PGRs and 3% sucrose remained viable (ratings of ≥1) for 21 months of storage in bags. Storage on reduced nitrogen (MS with 25% nitrogen), PGRs, and 3% sucrose kept ‘Grushovka Vernenskaya’ shoot condition rated >2 at 21 months. Addition of 0.5 or 1 mg⁻¹ abscisic acid (ABA) also improved plant ratings at 21 months. The longest storage for ‘Grushovka Vernenskaya’ was 33–39 months with PGRs and 3% sucrose in either tubes or jars. Addition of abscisic acid (ABA) to the medium did not improve storage of plantlets in jars and tubes at 15 months. TM-6 stored best in tubes on 3% sucrose with PGRs or in jars on 2% mannitol and 2% sucrose. Overall it appears that cold storage of apple shoot cultures can be successful for 21 months in tissue culture bags with 25% MS nitrate nitrogen, 3% sucrose, and no PGRs or for 33 months in jars or tubes on MS with 3% sucrose and PGRs. Preliminary RAPD analysis found no significant differences between plants stored for 39 months and non-stored controls.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Efficient plant regeneration from cell cultures of ornamental statice, Limonium sinuatum mill.

Summary A plant regeneration system from cell suspension cultures was established in an important ornamental crop, Limonium sinuatum Mill. cv. ‘Early Rose’. Friable callus was initially induced from leaf segments of in vitro-cultured seedlings on 0.25% gellan gum-solidified half-strength Murashige and Skoog [1/2MS] medium containing 1.0 mg l⁻¹ (4.14 μM) picloram. These calluses were maintained as cell suspension cultures, which showed high proliferation ability with about 80 times increase in fresh weight during the 2-wk interval of subculture. Shoot regeneration from these cell cultures was achieved by cytokinins, especially zeatin, which was the most effective in producing normal shoots with reduced hyperhydration when used in combination with 0.5% gellan gum. Shoot regeneration ability was different among the cell lines originated from each different seedling. Shoot formation was observed at different frequencies on four of five cell lines whereas one cell line showed no shoot differentiation. Regenerated shoots detached from callus readily rooted 1 mo. after the transfer onto 0.5% gellan gum-solidified 1/2MS medium lacking plant growth regulators. The plantets were successfully transferred to the greenhouse after acclimatization. No ploidy changes were observed in the callus induced or in the regenerated plantlets. The regenerated plantlets that were transferred to the greenhouse after acclimatization grew normally and did not any morphological signs of somaclonal variation.     

Direct shoot and cormlet regeneration from leaf explants of ‘Silk’ banana (AAB)

Summary Direct shoot and cormlet regeneration from leaf explants were obtained in triploid dessert banana cultivar Nanjanagud Rasabale (NR) that is classified under the group ‘Silk’ and has the genotype AAB. The response for both cormlet and direct shool formation was observed only in leaf explants obtained from shoots cultured in liquid medium but not in similar explants obtained from shoots grown on gelled medium. Shoot initiation occurred after a sequential culture of leaf (sheath) explants on modified Murashige and Skoog (MS) medium supplemented with different growth regulators. In the sequence, the leaf explants were cultured first on medium with a high level (22.4 μM) of benzyladenine (BA), second on indolc-3-butyric acid (IBA) supplemented medium, and third on reduced BA medium under incubation in the dark. The highest adventitious shoot regeneration in 24% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants incubated at the first step in medium with 22.4 and 0.198 μM IBA. Further growth and complete shoot formation occurred under incubation in a 16-h photoperiod. While keeping the culture conditions constant and replacing BA with picloram (0.83–20.71 μM) in the initial step, adventious origin of cormlets occurred in 12% of the explants. However, when rhizome explants (also obtained from shoots grown in liquid medium) were cultured with various growth regulators in the first step, medium containing 2,4,5-trichlorophenoxyacctic acid (7.82 μM) produced friable callus that re-differentiated into roots only. Physical forms of the medium, ie.e. agar-gelled or liquid, imparted specific effects on the extent of multiplication of leaf-regenerated shoots with no differences in morphology and growth patterns when compared to those of meristem-derived plants.     

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.     

Influence of cytokinin levels on <Emphasis Type="Italic">in vitro</Emphasis> propagation of shy suckering chrysanthemum “Arka Swarna” and activation of endophytic bacteria

In an effort to develop a sustainable protocol for the micropropagation of a shy suckering elite chrysanthemum cv. Arka Swarna (yellow pompon type), in vitro cultures were established using surface-sterilized nodal microcuttings (1–1.5 cm) from polyhouse-grown plants on MS medium containing 3% sucrose, 0.25% phytagel, and 5 μM benzyl adenine (BA) or kinetin. Microbial contamination in the range of 6–24% was encountered during the first in vitro passage. Apparently clean cultures after one passage on MS basal medium were transferred to medium with BA or kinetin (0, 1, 5, 10, or 20 μM) in culture bottles, and were monitored for eight in vitro passages (1 mo. each) for growth and microbial contamination. Plant growth regulator (PGR)-free medium was the best for sustainable micropropagation over successive in vitro passages yielding a single shoot from cultured microcuttings. Higher cytokinin levels inhibited rooting and induced one or more shorter shoots with close nodes resulting in low propagation rates. All apparently clean stocks revealed covert endophytic bacteria during tissue-indexing using bacteriological media. Three distinct bacterial morphotypes were isolated from such stocks, identified based on 16S rRNA gene sequence analysis as different morphotypes of Curtobacterium citreum. The endophytes tended to show obvious growth on chrysanthemum culture medium with increase in cytokinin levels (5–20 μM), but such growth was not noticed in inoculations on MS medium without plants. Sustainable micropropagation of cv. Arka Swarna for more than 2 yr with the resident endophytic bacteria in covert form was realized on PGR-free MS medium giving a net propagation rate of three to four times over a subculture cycle of 2–3 wk.     

A simple and rapid procedure to establish embryogenic cell suspensions as a source of protoplasts for efficient plant regeneration from two Chinese commercial rice cultivars

An efficient and rapid procedure has been developed to establish embryogenic cell suspension cultures of two Japonica Chinese commercial rice cultivars, Zhonghua 8 and Eryi 105. Embryogenic cell suspensions of both varieties were established from 0.5–1.0 g fresh weight of embryogenic callus in AA medium within 2.5 months of the initiation of callus from sterilised seeds. The previously reported subculture of callus on semi-solid medium for 4–8 weeks prior to transfer into liquid medium was unnecessary and caused delay in the establishment of embryogenic cell suspensions. Protoplasts were isolated reproducibly from cell suspensions up to 18 months after their initiation, with protoplast plating efficiencies attaining 0.15–0.37%. Reproducible plant regeneration from 14–26% of the protoplast-derived tissues was achieved without the requirement for nurse cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

The influence of Bipolaris sorokiniana and Drechslera dictyoides metabolites on cell membrane permeability of Festuca pratensis callus

The aim of this study was the evaluation of membrane permeability of callus cells of several Polish meadow fescue cultivars, which were treated with toxins of two leaf spot pathogens Bipolaris sorokiniana and Drechslera dictyoides. Fungus metabolites were obtained by the method described by Lepoivre et al. (1986). Calli of cultivars ‘Skrzeszowicka’, ‘Skawa’, ‘Westa’, POB 282, POB 383, KOA 186 have been selected on medium with metabolites for two weeks. Next the conductivity test of electrolyte leakage and of total ion contents in the examined tissue was done. On the base of this data the membrane permeability coefficients for each cultivar were calculated. Toxins of B. sorokiniana damaged the cell membranes more strongly than metabolites of D. dictyoides. The significant differences of several objects sensitivity to the influence of B. sorokiniana metabolites were stated. These differences were not observed in the case of the influence of D. dictyoides metabolites on the examined tissue.     

Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya

This study establishes the widespread prevalence of fastidious or viable but non-culturable endophytic bacteria in field shoots and in unsuspicious shoot-tip cultures of papaya (Carica papaya L.) against the norm of asepsis in vitro. A total of 150 shoot-tips (approximately 10 mm) were inoculated on MS-based culture medium after surface sterilization of field-derived axillary shoots of cv. Surya during November or January (100 and 50, respectively) when 35–50% cultures showed endophytic microbial growth on culture medium. Indexing of apparently clean cultures using bacteriological media helped in detecting and removing additional 14–17% stocks with covert bacteria during the first two passages. The rest of the stocks stayed consistently index-negative during the first eight subculture cycles, but appeared positive in PCR-screening undertaken thereafter employing universal bacterial 16S rRNA gene primers indicating the association of non-cultivable bacteria. Direct sequencing of the PCR product yielded overlapping nucleotide data signifying mixed template or the presence of diverse endophytic microorganisms. This was confirmed by light microscopy of tissue sap revealing viable bacteria in considerable numbers, which were detected under phase contrast or with negative staining. Planting tissue segments or applying homogenate from these stocks on diverse bacteriological media did not induce the organisms to grow in vitro. The shoot cultures displayed variation in growth and rooting potential, the onus of such variation was solely attributable to the associated microorganisms. The findings were confirmed with additional field shoots and fresh in vitro stocks established subsequently. The observations have implications in micropropagation and all other applications involving plant cell, tissue, organ, and protoplast culture.     

Steroidal glycoalkaloid content of potato,tomato and their somatic hybrids

Summary Analyses of leaves and ‘tubers’ from somatic hybrids of potato and tomato (‘pomato’ with plastids of potato, ‘topato’ with plastids of tomato) produced by fusion of protoplasts from liquid cultures of dihaploid potato and mesophyll of tomato revealed the presence of the two major potato glycoalkaloids (α-solanine and α-chaconine) as well as the tomato glycoalkaloid (αtomatine). The total alkaloid content of leaves was greater than that of ‘tubers’ and similar to levels in the foliage of parent plants. However, glycoalkaloids were more abundant in hybrid ‘tubers’ than in normal potato tubers by a factor of 5–15. In hybrid foliage, approximately 98% of the alkaloid present was of potato origin whereas in ‘tubers’ the reverse was the case, with tomatine comprising 60–70% of the total alkaloid. The similarities in alkaloid content and ratios between the pomato and the topato lines indicate that plastomes do not influence the biosynthesis and distribution of these alkaloids. The results indicate that major secondary metabolites may prove useful for assessing the hybrid nature of such plants.