Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro

Efficacy of phloroglucinol in promoting growth and development of in vitro-derived shoot tips was studied in six potato (Solanum tuberosum L.) genotypes. Different concentrations of phloroglucinol (0, 0.08, 0.4, 0.8, 1.2 and 1.6 mM) were tested in combination with either 0.1 or 0.2 M sucrose in shoot tip proliferation medium based on MS (Murashige and Skoog, 1962) medium supplemented with 5.8 μM GA₃ (gibberellic acid), 1.1 μM BA (N⁶-benzyladenine) and 8.39 μM D-calcium pantothenate. Phloroglucinol fostered multiple shoot formation, promoted axillary shoot proliferation in terms of shoot tip fresh weight and shoot length, and stimulated root formation on the shoot tips. There was significant phloroglucinol × sucrose interaction for number of shoots developed per shoot tip, shoot tip fresh weight and number of roots induced per shoot tip. The beneficial effect of phloroglucinol on shoot tip survival was conspicuous only in genotypes that showed poor survival in the control proliferation medium. There were significant differences in response between the two sucrose levels with regard to shoot tip fresh weight and number of roots per shoot tip. Phloroglucinol in combination with 0.2 M sucrose induced maximum number of roots per shoot tip. Optimum shoot tip growth was fostered in medium containing 0.8 mM phloroglucinol and 0.2 M sucrose. High frequency multiple shoot formation in this medium ensures a faster rate of potato shoot tip multiplication within a limited time and space. This revised version was published online in June 2006 with corrections to the Cover Date.     

Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (<Emphasis Type="Italic">Pinus virginiana</Emphasis> Mill.)

Plant tissue necrosis and subsequent cell death are usually observed during in vitro regeneration in conifers, especially in plant regeneration via somatic organogenesis in pine species. Cell death is correlated with the elevated levels of peroxides. In this investigation, the effects of antioxidants on in vitro regeneration of Virginia pine (Pinus virginiana Mill.) were evaluated. Antioxidants, polyvinylpolypyrrolidone (PVPP) and 1,4-dithio-dl-threitol (DTT), were found to improve callus formation, shoot differentiation and growth, and shoot rooting by inhibiting tissue necrosis during the initiation of cultures and subculture of shoots. These treatments enabled the recovery and regeneration plants at high frequency through somatic organogenesis. Compared to the control, the frequencies of callus formation, shoot growth, and shoot rooting increased 15, 26, and 19%, respectively, by addition of 5 g/l PVPP and 2 g/l DTT. Higher peroxidase activity of tissue cultures during subculture from callus proliferation medium to shoot differentiation medium and to rooting medium was observed. The addition of antioxidants reduces and inhibits browning by reducing the accumulation of peroxidase.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - DTT 1,4-Dithio-dl-threitol - IBA Indole butyric acid - NAA -Naphthaleneacetic acid - PVPP Polyvinylpolypyrrolidone     

High-frequency induction of adventitious shoots from hypocotyl segments ofLiquidambar styracifiua L. by thidiazuron

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.Abbreviations BA Benzyladenine - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyacetic acid     

The effect of transforming growth factor-β on fibroblast cell proliferation in intact connective tissue in vitro

Summary Transforming growth factor-β (TGF-β) has varying effects on cell proliferation, stimulating some cell types while inhibiting others. Its effect on proliferation has mostly been assessed in cell cultures without consideration for the influence of a tissue matrix. In the present investigation we studied the effect of TGF-β on fibroblast cell proliferation in intact connective tissue in vitro using the membranous part of the rat mesentery. Mesenteric membranes were spread over the hole of a cytocentrifuge paper, incubated in vitro, and exposed to various concentrations of TGF-β with or without serum added. At designated times after incubation, the specimens were fixed, spread out on microscope slides, and stained by the Feulgen reaction. Cell proliferation was estimated by counting mitoses in fibroblasts and mesothelial cells and by DNA cytometry of fibroblast nuclei using computer assisted image analyses. Higher concentrations of TGF-β significantly increased proliferation estimated as either the percentage of cells in the S+G₂ phase of the cell cycle or the mitotic index when serum was added. In medium without serum, TGF-β did slightly, but not significantly, increase proliferation. The results show that TGF-β stimulates connective tissue cell proliferation dose-dependently in intact connective tissue in vitro and that addition of serum to the medium is a prerequisite for optimal stimulation.     

Microcutting leaf area,weight and position on the stock shoot influence root vigour,shoot growth and incidence of shoot tip necrosis in grape plantlets in vitro

The physiological parameters of microcuttings, namely leaf area, weight (wt) and position on the stock shoot, had significant effects on root vigour, microshoot growth and incidence of shoot tip necrosis (STN) in cultures of grape (Vitis vinifera L.) ‘Arka Neelamani’. Single-node leafy cuttings cultured in MS medium containing 1 μM IAA and 0.1 μM GA₃ generally rooted first and subsequently sprouted into a single shoot. Small leafed cuttings exhibited slow root emergence, poor quality roots, early sprouting and weak shoot growth. Large leafed cuttings on the other hand, showed early rooting, vigorous roots, delayed sprouting and healthier shoots. Significant correlations were observed between fresh root wt per plantlet at 1 month in culture and wt of lamina, total wt and leaf area of the cuttings in that order. A significant correlation also existed between wt of roots and height or wt of the sprout that developed. The study suggests that the weight or area of the leaf governed the root growth in a microcutting. STN was observed in some plantlets particularly those derived from large leafed cuttings. Such cuttings showed vigorous roots and delayed but fast sprout growth that ended in STN. Plantlets showing STN had less Ca⁺⁺ and Mg⁺⁺ in the shoot tissue than in the shoots of normal plantlets while the roots showed similar Ca⁺⁺ or higher Mg⁺⁺ contents. Both had comparable amounts of cytokinins in shoot and root tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of photoperiod,mineral medium strength,inorganic ammonium,sucrose and cytokinin on root,shoot and microtuber development in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams

The effects of photoperiod (8, 12 or 16 h), mineral medium strength (dilutions of a tuberization medium, the T medium), sucrose (0, 2, 4, 8% w/v) and kinetin (0, 2.5μM) on the development of roots, shoots and microtubers in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams were evaluated. All of the factors were found to have substantial effects on microtuber induction in these two species. The effects of high and low inorganic ammonium containing media on microtuberization of yam shoot cultures indicated that ammonium ions inhibited microtuber induction in D. alata but not in D. bulbifera. Microtubers of D. alata were only formed on shoot cultures if these were held under 8-h days. D. bulbifera cultures on the other hand produced microtubers under this photoperiod treatment as well as under 16-h photoperiods provided that kinetin was present in media at 2.5μM. Most microtuberization in D. alata shoot cultures occurred on full-strength T medium supplemented with 2% sucrose, 2.5μM kinetin held under 8-h photoperiod at 25°C, whereas most microtuberization in D. bulbifera shoot cultures occurred on full-strength MS medium supplemented with 4% sucrose, 2.5μM kinetin held under 8-h photoperiods at 25°C. Under these two sets of conditions, yam shoot cultures consistently produced microtubers with individual weights in excess of 100 mg which were large enough to be capable of direct planting and subsequent growth in unsterilized soils.     

Effect of silver nitrate on multiple shoot formation of Virginia-type peanut through shoot tip culture

Summary A protocol for micropropagation of Virginia-type peanut plants, an ancient crop of the New World, is reported. This study was conducted to explore the effect of silver nitrate (AgNO₃), alone or in combination with growth regulators, on multiple shoot formation from shoot tip culture. Incorporation of AgNO₃ into the medium, without growth regulators, induced regeneration of the explants (which did not develop at all in the AgNO₃-free medium), and stimulated the emergence of axillary shoots. When AgNO₃ was added in combination with cytokinins and α-naphthaleneacetic acid (NAA), maximum average shoot number per regenerating explant was recorded (6.3) in Murashige and Skoog (MS) medium containing 33 μM 6-benzyladenine, 5.3 μM NAA, and 23.54 μM AgNO₃. Moreover, AgNO₃ showed a positive and marked effect on both shoot elongation and the reduction of callus proliferation from the basal ends of shoot tips. Following a period of elongation, the shoots were rooted in hormone-free Ms medium, showing no residual effects due to the long-term culture in AgNO₃-containing media. Acclimatization was easily obtained after plantlets were transferred to pots under greenhouse conditions, with 90% survival.     

Ethylene and CO2 affect direct shoot regeneration from the petiolar ends of Paulownia kawakamii leaves cultured in vitro

The effects of ethylene and CO₂ on shoot regeneration in excised leaf cultures of Paulownia kawakamii were examined. When both the gases were prevented from accumulating in the headspace of cultures using mercuric perchlorate and potassium hydroxide traps, shoot regeneration frequency improved and callus production was reduced compared to the control and cultures with only one of the gases trapped. Incorporation of either aminoethoxyvinylglycine (AVG) or 1-amino-cyclopropane-1-carboxylic acid (ACC) in the culture medium caused significant reduction in shoot regeneration. There was profuse callus production in the presence of high amounts of ACC, which was accompanied by over sixfold increase in the rate of ethylene production. However, in the presence of AVG callus production was delayed and shoot regeneration decreased, suggesting that low levels of ethylene might be needed for de novo shoot bud induction in Paulownia cultures.Abbreviations IAA Indole-3-acetic acid - MP mercuric perchlorate - AVG aminoethoxyvinylglycine - ACC 1-aminocyclopropane-1-carboxylic acid     

A rapid in vitro propagation protocol for Piper barberi Gamble, a critically endangered plant

Summary A protocol is described for rapid multiplication of Piper barberi Gamble (Piperaceae) through shoot tip and nodal explant cultures. Nodal explants with a single axillary meristem showed three times better response with respect to shoot proliferation when compared to shoot tip explants. The best shoot proliferation response of nodal explants was observed with a cytokinin combination of N₆-benzyladenine (4.43 μM) and kinetin (2.32 μM), with 88% bud break. The number of shoot initials (2.4) produced per nodal explant was twice the number of shoot initials (1.2) per shoot tip. An average of 6.9±0.58 adventitious shoots were observed from the proximal end of the internodal explants on Mursashige and Skoog (1962) (Ms) basal medium supplemented with N₆-benzyladenine (2.22 μM) and kinetin (0.46 μM). A multiplication rate of 82 shoots per explant could be achieved after 9 wk of subculturing. The in vitro shoots were rooted on one-half and one-quarter MS basal medium. The shoots rooted on one-quarter MS in the dark produced eight roots with an average root length of 3.36 cm and 98% survival. These plants were transferred to the field with a survival rate of 75%.     

Nutrient utilization in liquid/membrane system for watermelon micropropagation

Watermelon [Citrullus lanatus (Thunberg) Matsumura and Nakai] proliferating shoot meristems from established shoot cultures were inoculated on modified Murashige and Skoog salts medium supplemented with 10 μM 6-benzyladenine (BA) for shoot proliferation and on similar medium supplemented with 1 μM BA and 10 μM gibberellic acid (GA₃) for shoot elongation. Agar-solidified medium and microporous polypropylene membrane rafts in liquid medium were used to support the tissues. Growth over culture time of proliferating and elongating tissues in liquid and agar-solidified media were compared. Nutrient depletion in liquid medium was monitored and quantified using ion selective electrodes. Tissue fresh weights in both proliferation and shoot elongation media were greater in liquid than in agar-solidified medium. Relative dry matter content, however, was greater in agar-solidified than in liquid medium. More shoots elongated in agar-solidified than in liquid medium. The numbers of buds or unelongated shoot meristems, however, were comparable for both the liquid and agar-solidified medium. Proliferating and elongating tissues in liquid medium used Ca⁺⁺ and K⁺ minimally. NO ₃ ⁻ was utilized but not depleted by proliferating tissues. NH ₄ ⁺ , however, was depleted. Most of the NH ₄ ⁺ was utilized by the proliferating tissues within 21 days of culture when growth rate was greatest. At 35 days, residual Ca⁺⁺, K⁺, NO ₃ ⁻ , and NH ₄ ⁺ in proliferation medium were 81.0%, 67.8%, 55.7%, and 1.2% of initial levels, respectively. NO ₃ ⁻ and NH ₄ ⁺ in shoot elongation medium were depleted. The greatest NO ₃ ⁻ and NH ₄ ⁺ utilization was observed during the first 14 days of culture when the largest growth rate was obtained. The residual Ca⁺⁺, K⁺, NO ₃ ⁻ , and NH ₄ ⁺ in shoot elongation medium at 38 days were 63.5%, 37.9%, 21.2%, and 24.3% of initial concentrations, respectively. At the end of experiment, 72.3% and 42.8% of initial sugars were still remaining in the shoot proliferation and shoot elongation medium, respectively. Technical Contribution No. 3236 of the South Carolina Agricultural Experiment Station.     

Influence of carbon source on shoot multiplication and adventitious bud regeneration in in vitro beech cultures

Experiments were performed to determine the effects ofcarbon source and concentration on shootmultiplication in shoot cultures of Fagussylvatica (one clone) and F. orientalis (twoclones) and on the induction of adventitious shootbuds from leaf and internode explants of F.orientalis. In general, glucose was the best carbonsource for both axillary branching and adventitiousshoot regeneration. Shoot-tip explants grown on 3–4%glucose medium produced more shoots than those onsucrose or fructose. For maximum shoot length, glucosemedium was best for two of the three clones, and 4%sucrose for the other. The number of shoots was theparameter most influenced by glucose concentration inthe adventitious shoot regeneration experiments, thenumber increasing with sugar concentration. The lowesthyperhydricity rate occurred in the presence ofsucrose in both species. Shoot growth and quality wasnegatively affected by fructose supplied media. Theuse of filter-sterilized rather than autoclavedfructose neither stimulated shoot growth nor reducedthe incidence of hyperhydricity in all three clones.The response of shoot cultures to differentcarbohydrate treatments appears to some extent to begenotype dependent.     

Nitrogen amendments reduce the growth of extramatrical ectomycorrhizal mycelium

The effect of three different nitrogen sources on the growth of external ectomycorrhizal mycelium was studied in Perspex micorocosms. Nonsterile peat was used as substrate. Five different fungal isolates growing in symbiosis with pine seedlings were investigated: two isolates of Paxillus involutus, one of Suillus bovinus and two unidentified ectomycorrhizal fungi isolated from ectomycorrhizal root tips. Three different nitrogen sources were used: ammonium as (NH₄)₂SO₄, nitrate as NaNO₃ and a complete nutrient solution (Ingestad 1979), and three different nitrogen concentrations, 1, 2 or 4 mg N/g dry wt. of peat. The mycelial growth of all fungi was found to be negatively affected by the nitrogen amendments, although the sensitivity to nitrogen varied between the isolates. One of the unidentified isolates was extremely sensitive and growth was completely inhibited by all nitrogen treatments. In contrast, the growth of one of the P. involutus isolates was only slightly reduced by the nitrogen amendments. The different nitrogen sources all reduced growth, and since no significant difference was found between the nitrogen sources or between the different nitrogen concentrations the results were pooled to give one value that summarized the effect of nitrogen on mycelial growth. Thus, the mycelial growth of one of the two P. involutus isolates was reduced to approximately 80% of the growth in the control, the other P. involutus and one of the unidentified fungi, vgk 2 89.10, were reduced to 40–50% of the control growth, S. bovinus to 30% of the control and the most sensitive fungus, the unidentified isolate vg 1 87.10, was reduced to 3% of the growth in the control treatment. In all experiments, the shoot to root ratio generally increased, mainly as a result of increased shoot growth.     

In vitro Storage of Strawberry and Raspberry in Calcium-Alginate Beads at 4°C

Three-millimeter-long shoot tips of strawberry 'Senga Sengana' and raspberry 'Norna' encapsulated in calcium alginate were stored in vitro at 4 °C in the dark. The cultures which were donors for the shoot tips were grown before encapsulation on shoot multiplication media (Boxus medium with 2.2 µM BAP and 2.46 µM IBA for strawberry, and MS medium with NH₄NO₃ and KNO₃ reduced by 50%, and with 3.55 µM BAP and 0.49 µM IBA for raspberry) as well as on these media supplemented with 10 g l^–1 mannitol or paclobutrazol (1.7 µM for strawberry and 3.4 µM for raspberry). Sodium alginate was dissolved in water, water with sugar or in a culture medium without growth regulators. Regrowth ability of the stored explants and in vitro multiplication in three successive subcultures were evaluated. The encapsulated shoot tips could be stored for 9 months in beads containing sugar or a culture medium. The pre-conditioning of the donor cultures on a mannitol containing medium was beneficial for regrowth ability. The multiplication rate of strawberry and raspberry shoots in the first subculture after storage was lower than that of non-stored cultures. Particularly low multiplication was obtained for strawberry which had been stored for 9 months and for raspberry stored for 3 and 6 months, in combinations where the beads were prepared by dissolving sodium alginate in water. Multiplication of strawberry in the second subculture was generally higher than in non-stored cultures, but multiplication of raspberry was lower also in the second subculture, with the exception of the combination stored for 9 months and pre-cultured on mannitol. In the third subculture, shoot multiplication in both species was similar to that in non-stored cultures.     

Effects of some non-pathogenic micro-organisms on the growth of gnotobiotic barley plants

Summary Mated cultures of Blakeslea trispora, which produce trisporic acid, inhibited root and shoot extension of barley plants grown in gnotobiotic cultures, where the carbon from roots was the sole source of carbon available to the microorganism. Unmated cultures of B. trispora and Mucor hiemalis, which do not synthesize trisporic acid, inhibited root extension only. Azotobacter chroococcum had a variable effect in different experiments on both root and shoot extension but could at least partially relieve the inhibitory effect of B. trispora on root growth. Plant growth regulators, applied to roots exogenously, produced effects on plants which, although comparable with some of the inhibitory effects of the micro-organisms, were not comparable with the stimulatory effects of A. chroococcum. re]19751127     

Alterations in growth and crassulacean acid metabolism (CAM) activity of in vitro cultured cactus

Unlike C-3 plants, cacti possess a crassulacean acid metabolism (CAM) physiology that can alter the pattern of carbon uptake and affect plant growth under artificial environmental conditions, especially in tissue culture. In vitro-derived plantlets of Coryphantha minima grew 7-fold larger than plants cultured under similar ex vitro conditions. Growth regulators incorporated into the culture media during shoot proliferation stage of micropropagation had a strong influence on this increased growth. Other important factors that contributed to increased growth under in vitro conditions were high relative humidity and sugar in the culture medium. An analysis of gas exchange and daily fluctuations of malic acid levels revealed an increase in net photosynthetic rate, in terms of carbon assimilation, by in vitro plants compared with that of ex vitro plants. This stimulated photosynthesis in the presence of an external carbon source was unexpected but apparently true for cacti exhibiting CAM physiology. Unlike CAM plants grown in ex vitro conditions, net CO₂ uptake by in vitro-cultured cacti occurred continuously in the light as well as the dark. Once regenerated, cacti were transferred to ex vitro conditions where the normal CAM pathway resumed with a concomitant reduction in growth and CO₂ uptake. These results showed that growth of cacti can be considerably accelerated by in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.     

Importance of seed Zn content for wheat growth on Zn-deficient soil

Eggplants (Solanum melongena L. cv. Bonica) were grown in a glasshouse during summer under natural light with one unbranched shoot or one shoot with 3 to 4 branches and with or without fruit in quartz sand buffered and not buffered with 0.5% CaCO₃ (w : v), respectively. Nutrient solutions supplied contained nitrate or ammonium as the sole nitrogen source. Compared with nutrient solutions containing nitrate (10 mM), solutions containing ammonium (10 mM) caused a decrease in net photosynthesis of eggplants during early stages of vegetative growth when grown in quartz sand not buffered with CaCO₃. The decrease was not observed before leaves showed interveinal chlorosis. In contrast, net photosynthesis after bloom at first increased more rapidly in eggplants supplied with ammonium than with nitrate nitrogen. However, even in this case, net photosynthesis decreased four weeks later when ammonium nutrition was continued. The decrease was accompanied by epinasty and interveinal chlorosis on the lower leaves and later by severe wilting, leaf drop, stem lesions, and hampered growth of stems, roots, and fruits. These symptoms appeared later on plants not bearing fruits than on plants bearing fruits. If nutrient solutions containing increasing concentrations of ammonium (0.5–30 mM) were supplied after the time of first fruit ripening, shoot growth and set of later flowers and fruits were promoted. In contrast, vegetative growth and reproduction was only slightly affected by increasing the concentration of nitrate in the nutrient solutions. In quartz sand buffered with CaCO₃ ammonium nutrition caused deleterious effects only under low light conditions (shade) and on young plants during rapid fruit growth. If eggplants were supplied with ammonium nitrogen before bloom, vegetative growth was promoted, and set of flowers and fruit occurred earlier than on plants supplied with nitrate. Furthermore, the number of flowers and fruit yield increased. These effects of ammonium nutrition were more pronounced when plants were grown with branched shoots than with unbranched shoots. The results indicate that vegetative and reproductive growth of eggplants may be manipulated without causing injury to the plants by supplying ammonium nitrogen as long as the age of the plants, carbohydrate reserves of the roots, quantity of ammonium nitrogen supplied, and pH of the growth medium are favourable. T W Rufty Section editor     

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA₃ (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro Micrografting of Mature Pistachio (Pistacia vera var. Siirt)

The success of various in vitro micrografting methods of shoot tips of pistachio (Pistacia vera L. var. Siirt) have been examined. Excised zygotic embryos that germinated in vitro were used as rootstocks. Current year shoot tips from mature trees of pistachio micrografted onto in vitro juvenile rootstocks, resulted in the restoration of shoot-bud proliferation. Variables tested include a size of microscion, grafting method, effects of culture medium and effects of time of the year at which shoot tips were used. The results indicate that the easiest and most successful method for grafting was slit micrografting. High levels of micrograft take were achieved with 2–4 mm (56.75%) and 4–6 mm (79.25%) long scions obtained from the regenerated shoot tips. The survival rate of the shoot tips was directly related to time of the year. The best growth of microscion was obtained with the in vitro forced shoot tips rather than with shoot tips excised from tree. Slow growth and lack of axillary shoot development on the micrografts was noticeable when the micrografts were cultured on hormone-free and germination medium. In vitro micrografted plantlets were successfully weaned and no problems were encountered with the establishment of micrografted plants in vivo.     

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.