A Novel screening method of cellulase-producing bacteria based on <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> var. <Emphasis Type="Italic">nicotianae</Emphasis>

Cellulase is the key to utilize the renewable and abundant cellulose resource, cellulase-producing microorganism is an important source of cellulase. The traditional screening method of cellulase-producing microorganism is low efficacy and not macroscopic. The screening method in this study was based on the interactive culture character between cellulase-producing bacteria and Phytophthora parasitica var. nicotianae on plates, the results indicated that the inhibition zone and cellulase activity of bacterial strains are conformity on the whole, so the screening method is very quickly and apparent     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Propagation of <Emphasis Type="Italic">Haemaria discolor</Emphasis> via <Emphasis Type="Italic">in vitro</Emphasis> seed germination

In vitro propagation protocol for Haemaria discolor (Ker) Lindl. var. dawsoniana by artificial cross-pollination and asymbiotic germination of seeds has been developed. Fruit set (100 %) was obtained when the pollinia and ovules of various aged flowers were used for pollination. In vitro germination of seeds obtained from capsules of various ages was achieved on half-strength Murashige and Skoog’s (MS) medium supplemented with 3 % sucrose and 0.85 % agar. The germinated seedlings were cultured on half-strength MS medium with 0.2 % activated charcoal, 8 % banana homogenate, 0.1 mg dm⁻³ 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) and 1 mg dm⁻³ α-naphthaleneacetic acid (NAA). Ninety-six percent of plantlets survived after hardening in greenhouse.This research was supported by grant (91AS-3.1.1-CI-C3) from the Council of Agriculture, Executive Yuan of Taiwan and grants (NSC-89-2317-B055-002 and NSC-91-2317-B324-001) from the National Science Council of Taiwan. This paper is Agricultural Research Institute Contribution No. 2158.     

<Emphasis Type="Italic">In vitro</Emphasis> minimum growth for conservation of <Emphasis Type="Italic">Drosophyllum lusitanicum</Emphasis>

The present paper reports a protocol for minimum growth conservation of Drosophyllum lusitanicum (L.) Link. in vitro. Double-node cuttings were maintained for 4, 8 and 12 months at 5 or 25 °C in the dark. The effects of sucrose either alone at 5, 20, 30, 40 and 60 g dm⁻³ or at 20, 40 and 60 g dm⁻³ in combination with 20 g dm⁻³ mannitol, on survival and post-storage shoot multiplication efficiency were investigated. The cultures could effectively be conserved under minimum growth at 5 °C for 8 months on Murashige and Skoog’s medium supplemented with 60 g dm⁻³ sucrose, 20 g dm⁻³ mannitol and 0.91 μM zeatin. Following extended conservation, the cultures could be successfully regenerated into new shoots, and they were morphologically similar to those of non-stored controls.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Pelecyphora aselliformis</Emphasis> erhenberg (cactaceae)

Summary In vitro propagation of Pelecyphora aselliformis, a Mexican cactus which is considered rare and is highly valued in the commercial market, was initiated using seeds as explants. The longitudinal explants from seedlings germinated in vitro were cultivated on Murashige and Skoog medium containing 8.8 μM benzyladenine (BA) or 4.6 μM kinetin at pH 7.0. After 120 d, each explant gave rise to five shoots and this number of shoots increased 20–25% after subculture. The hyperhydricity was similar in both media, but callus formation was lower on the medium with BA. The shoot development, in terms of epicotyl length, and fresh and dry weight after 6 wk, was also recorded. The epicotyl length was similar on shoot-forming media but the quality of shoots was better on media containing BA. In about 1 yr, 500–600 well-defined shoots were obtained. The rooting of shoots was very slow and a vigorous radical system was observed after 1 yr of culture.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis>: influence of cytokinins

The role of cytokinins in the promotion of flowering in the endangered species Kniphofia leucocephala Baijnath. was investigated using shoots maintained in culture for 3 years. The highest percentage flowering (65%) was obtained on media containing 20 μM benzyladenine (BA). The inclusion of isopentenyladenine and zeatin in the media also resulted in flowering, but these treatments were less effective than BA in inducing flowering. The effect of cytokinins on flowering was dose-dependent, with high concentrations of BA inhibiting flower formation. Treatments that resulted in rooting of explants produced no flowers. The resulting inflorescences in all treatments did not mature and senesced prematurely, even when gibberellic acid (GA₃) was applied post-flower-emergence.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation and cryostorage of <Emphasis Type="Italic">Syzygium francissi (Myrtaceae)</Emphasis> by the encapsulation-dehydration method

Summary An efficient procedure for the in vitro propagation and cryogenic conservation of Syzygium francissi was developed. The maximum number of shoots per explant was obtained on a Murashige and Skoog (MS) medium supplemented with 4.5 μM benzyladenine and 0.5 μM indole-3-butyric acid (IBA). The in vitro-propagated shoots produced roots when transferred to MS medium containing IBA, indold-3-acetic acid, or naphthaleneacetic acid at various concentrations. Rooted microshoots were transferred to a coco-peat, perlite, and vermiculite (1∶1∶1) mixture, and hardened off under greenhouse conditions. Ninety-five percent of rooted shoots successfully acclimatized in the greenhouse. Shoot tips excised from in vitro-grown plants were successfully cryostoraged at −196°C by the encapsulation-dehydration method. A preculture of formed beads on MS medium containing 0.75 M sucrose for 1 d, followed by 6 h dehydration (20% moisture content) led to the highest survival rate after cryostorage for 1h. This method is a promising technique for in vitro propagation and cryopreservation of shoot tips from in vitro-grown plantlets of S. francissi germplasm.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Direct shoot regeneration from immature inflorescence cultures of <Emphasis Type="Italic">Chlorophytum arundinaceum</Emphasis> and <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L⁻¹ BA, 150 mg L⁻¹ Ads, 0.1 mg L⁻¹ NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation. Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L⁻¹ IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement prograrmme.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Pisum sativum in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivin, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm⁻³ kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T₀ putative transformants and their seed progenies (T₁ to T₃ generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T₀ – T₃ plants were morphologically normal and fertile.This research was supported by the National Agency for Agricultural Research (grants No. QE 0046 and QF 3072) and Ministry of Education of the Czech Republic (grant No. ME 433).     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

Induction of streptomycin-resistant plantlets in <Emphasis Type="Italic">Solanum surattense</Emphasis> through <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis

The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.     

Rapid regeneration of <Emphasis Type="Italic">Phaseolus angustissimus</Emphasis> and <Emphasis Type="Italic">P. vulgaris</Emphasis> from very young zygotic embryos

A rapid regeneration protocol for proembryos of Phaseolus angustissimus as young as 1 day after pollination (DAP) involving pod culture for 1 week followed by embryo culture for 2 weeks and embryo germination for 1 or 2 weeks is provided. Optimization of the media was conducted with pods collected 3 DAP. The best pod culture medium was composed of basal medium [(Phillips and Collins 1979) salts with (Geerts et al. 2001) vitamins], 1000 mg l⁻¹ glutamine, 1000 mg l⁻¹ casein hydrolysate, 3% sucrose and 0.5% agar. Embryo culture medium consisted of basal medium with 500 mg l⁻¹ glutamine, 250 mg l⁻¹ casein hydrolysate, 1.9 μM ABA, 3% sucrose and 0.5% bacto-agar. Embryos developed into plantlets on germination medium containing basal medium with 0.25 μM BA, 3% sucrose and 0.7% bacto-agar. Fertile, normal plants were recovered from direct embryogenesis and from micrografted embryo-derived shoots. Embryos obtained from pods collected 3 DAP regenerated plantlets at a rate of 29.3%, while embryos from pods collected 2 DAP and 1 DAP regenerated at rates of 20.2 and 4%, respectively. A second accession of P. angustissimusregenerated at a rate of 26.2%. Using this 5-week protocol for P. vulgaris resulted in a plantlet regeneration rate of 12.5%.     

<Emphasis Type="Italic">In vitro</Emphasis> culture studies on <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

Summary Shoot apex, nodal, and leaf explants of Stevia rebaudiana Bertoni can regenerate shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 8.87 μM) and indole-3-acetic acid (5.71 μM). Rooting of the in vitro-derived shoots could be achieved following subculture onto auxin-containing medium. A survival rate of 70% was recorded at the hardening phase on the substrate cocopeat. The presence of the sweet diterpene glycosides, viz. stevioside and rebaudioside, was confirmed in the in vitro-derived tissues of Stevia using HPTLC techniques. Callus cultured on agar-solidified MS medium supplemented with BA (8.87 μM) and indole-3-butyric acid (9.80 μM) showed the highest sweetener content.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Acer grandidentatum</Emphasis> Nutt.

Bigtooth maple (Acer grandidentatum) is a promising ornamental tree that is not widely used in managed landscapes. Tissue culture has not been used successfully to propagate this taxon. We cultured single- and double-node explants from greenhouse-grown, 2-y old seedlings of bigtooth maples, which are indigenous to New Mexico, Texas, and Utah, on Murashige–Skoog (MS), Linsmaier–Skoog (LS), Driver–Kuniyuki Walnut (DKW), and Woody Plant (WPM) tissue culture media. Media affected shoot proliferation (P = 0.0242) but the zone of explant origin (P = 0.7594) did not. After four 30-d subcultures, explants on DKW media and WPM media produced 3.6 and 3.5 shoots per explant, respectively. Sprouting rates were highest on DKW, making DKW the best overall media for shoot proliferation. Double-node microshoots were rooted in vitro on DKW containing indole acetic acid (IAA). Microshoots represented six genotypes from three locations within Texas and New Mexico. Rooting percentage increased up to 15% as IAA concentration increased (P = 0.0040). There was 100% survival of rooted microshoots in vented Phytatrays containing one perlite: one peat moss (v/v). We conclude that DKW can be used to proliferate microshoots, and IAA induces rooting in microshoots of bigtooth maple.     

In vitro screening of mulberry (<Emphasis Type="Italic">Morus</Emphasis> spp.) for salinity tolerance

An efficient in vitro screening method has been developed for mulberry (Morus spp. ) to screen salinity-tolerant genotypes from a large population. Axillary buds from field-grown plants were cultured on MS medium containing five different concentrations (0.0%, 0.25%, 0.5%, 0.75% and 1.00%) of sodium chloride (NaCl) in order to study the shoot growth pattern. Rooting was also tested at four different concentrations of NaCl (0.0%, 0.1%, 0.2% and 0.3.%). NaCl has been found to inhibit the growth and development of mulberry shoots and roots in vitro. The survivability of the axillary buds of the genotypes tested was reduced from 83.7% for the controls to 6.1% in 1.0% NaCl. The average number of roots developed by the genotypes ranged from 11.9 (controls) to 0.2 (0.3% NaCl). Out of the 63 genotypes tested, only seven—Rotundiloba, English black, Kolitha-3, Berhampore-A, Kajli, BC₂59 and C776—developed roots in 0.3% NaCl. Root growth was also reduced drastically from 1.8 cm for the controls to 0.1 cm in 0.3% NaCl. To test the reproducibility of the results in soil, five tolerant and two susceptible genotypes, identified in this in vitro study, were selected and tested under ex vitro conditions. The significant correlation coefficients obtained between the performances of these genotypes under both types of cultural conditions revealed that in vitro screening of mulberry through axillary bud culture is an easy and efficient method to identify salt-adapted genotypes within a limited space and time period.Abbreviations BA 6-Benzylaminopurine - dS m^-1 Deci-Siemens per meter - EC Electric conductivity - NAA -Naphthaleneacetic acidCommunicated by G.C. Phillips     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation and alkaloids of <Emphasis Type="Italic">Hippeastrum vittatum</Emphasis>

Different concentrations of methyl jasmonate, spermine, casein hydrolysate, or progesterone combined with 16 mg/l 2 iP + 4 mg/l naphthalene acetic acid (NAA) were investigated in order to obtain higher multiplication rate and growth of Hippeastrum vittatum bulbs in vitro on MS basal medium. The highest multiplication rate (8.2 bulbs/explant) was attained with 80 mg/l spermine, while the highest bulb fresh weight (1.23 g/bulblet) was obtained with 4 mg/l methyl jasmonate. Progesterone at 20 mg/l or casein hydrolysate at 2.0 g/l gave the highest leaf length (14.1 and 13.2 cm, respectively). So, it can be advised to use 80 mg/l spermine combined with 16 mg/l 2 iP + 4 mg/l NAA to obtain the highest number of bulbs per explant with moderate leaf length and bulb fresh weight. Chemical analysis showed alternations in the alkaloid type ratio and number of compounds in the bulbs treated with methyl jasmonate (4 mg/l).