Arbuscular mycorrhizal fungi-parasite-host interaction for the control of<Emphasis Type="Italic"> Striga hermonthica</Emphasis> (Del.) Benth. in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

Five Glomus species (G. intraradices, G. albidum, G. mosseae, G. fasciculatum, and G. etunicatum) were compared against a check [without arbuscular mycorrhizal (AM) fungi, plus Striga] and control (without AM fungi or Striga) treatments for the control of Striga in a tolerant sorghum variety (War-wara bashi) in an experiment carried out in 12-cm-diameter clay pots. The experiment was carried out in a controlled growth chamber. G. mosseae significantly reduced the number of Striga emerging per plant, increased plant growth, shoot and total dry matter yield of sorghum, did not affect the root dry matter compared with the other AM fungi species, but had a comparable effect to the control treatment. All the AM fungi except G. mosseae, and also the Striga-infested treatment, increased the root:shoot ratio compared to the control treatment. The percent reduction (62%) of Striga emergence after G. mosseae inoculation resulted in about a 30% increase in total dry matter yield of sorghum over the control, while the total loss in dry matter yield of sorghum due to Striga infestation was 36%. Root colonization of sorghum by AM fungi was highest for G. mosseae (44%) followed by G. intraradices (24%) and G. albidum (23%) then G. fasciculatum (18%), with the lowest recorded for G. etunicatum (14%). No colonization of Striga roots was observed. The potential of AM fungi to reduce or to compensate for Striga infestation could be important for soil management, especially in the tropics, and for the reduction of Striga-resistant varieties of sorghum which are mycorrhiza-responsive.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Chrysanthemum</Emphasis> plantlets using light-emitting diodes

Effects of illumination spectrum on the morphogenesis of chrysanthemum plantlets (Chrysanthemum morifolium Ramat. ‘Ellen’) grown in vitro were studied using an illumination system consisting of four groups of light-emitting diodes (LEDs) in the following spectral regions: blue (450nm), red (640nm), red (660nm), and far-red (735nm). Taking into account all differences in shoot height, root length, and fresh and dry weight (FW and DW, respectively), observed while changing the total photon flux density (PFD), the optimal total PFD for growth of chrysanthemum plantlets in vitro was estimated. For 16 h photoperiod and typical fractions of the spectral components (14%, 50%, 28%, and 8%, respectively), the optimal total PFD was found to be 40 μmol m⁻² s⁻¹. Our study shows that the blue component in the illumination spectrum inhibits the plantlet extension and formation of roots and simultaneously increases the DW to FW ratio and content of photosynthetic pigments. We demonstrate photomorphogenetic effects in the blue region and its interaction with the fractional PFD of the far-red spectral component. Under constant fractional PFD of the blue component, the root number, length of roots and stems, and fresh weight of the plantlets have a correlated nonmonotonous dependence on the fractional PFD of the far-red component.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> regeneration of <Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.

A simple and efficient protocol for high frequency plant regeneration of a grain legume grasspea (Lathyrus sativus L.) is described. Of different explant types tested epicotyl segments were most responsive. Murashige and Skoog’s (1962) medium augmented with 17.76 µM 6-benzyladenine + 10.74 µM α-naphthaleneacetic acid showed the highest percentage of direct shoot regeneration. Among cultivars IC-120487 showed the highest regeneration frequency (80 %) with maximum shoot numbers (8.2 shoots per explant) and maximum average shoot length (4.1 cm). About 78 % of the regenerated shoots were rooted in half-strength MS medium containing 2.85 µM indole-3-acetic acid. After primary hardening the plantlets were established in soil with a survival rate of 75 %.     

Effect of mycorrhization on the accumulation of rishitin and solavetivone in potato plantlets challenged with<Emphasis Type="Italic"> Rhizoctonia solani</Emphasis>

The effect of colonization with the vesicular-arbuscular mycorrhizal fungus Glomus etunicatum on the content of rishitin and solavetivone was determined in potato plants cv. Goldrush challenged with Rhizoctonia solani. Mycorrhization stimulated significantly the accumulation of both phytoalexins in roots of plantlets challenged with R. solani but did not influence phytoalexin levels in non-challenged plantlet roots. No accumulation of solavetivone or rishitin was detected in shoots. In Petri dish bioassays, rishitin and solavetivone inhibited mycelial growth of R. solani.     

Enhanced multiplication and improved <Emphasis Type="Italic">ex vitro</Emphasis> acclimatization of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>

The proposed work describes a protocol for high-frequency in vitro regeneration through nodal segments and shoot tips in Decalepsis arayalpathra, a critically endangered medicinal liana of the Western Ghats. Nodal segments were more responsive than shoot tips in terms of shoot proliferation. Murashige and Skoog’s (MS) basal medium supplemented with 5.0 μM 6-benzyladenine (BA) was optimum for shoot initiation through both the explants. Among different combinations of plant growth regulators and growth additive screened, MS medium added with 5.0 μM BA + 0.5 μM indole-3-acetic acid + 20.0 μM adenine sulphate effectuated the highest response: 11.8 shoots per nodal segment and 5.5 shoots per shoot tip with mean shoot length of 9.2 and 4.8 cm, respectively. Half-strength MS medium with 2.5 μM α-naphthalene acetic acid was optimum for in vitro root induction. The plantlets with the well developed shoot and root were acclimatized in Soilrite? with 92 % survival rate in the field conditions. During acclimatization, chlorophyll content, net photosynthetic rate, stomatal conductance, and transpiration rate were gradually changed in dependence of formation of new leaves. Further, the changes in activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) as well as activity of carbonic anhydrase were also observed: a continuous rise in SOD activity, but a rise and fall in the activities of CAT, APX, and GR were also noticed. Maximum fresh mass (3.1 g plant^-1), dry mass (0.35 g plant^-1) of roots and 2-hydroxy-4-methoxybenzaldehyde content of 9.22 μg cm^-3(root extract) were recorded after 8 weeks of acclimatization.     

First report of <Emphasis Type="Italic">Verticillium tricorpus</Emphasis> isolated from potato tubers in Japan

In 1998, Verticillium sp. (CE98Vt1 and CE98Vt2) were isolated from discolored vascular structures of potato tubers sold at a market in Chiba Prefecture. These isolates were identified as Verticillium tricorpus on the basis of cultural and morphological characteristics and PCR diagnosis. This observed vascular discoloration of the potato tuber was demonstrated in three cultivars (Touya, Toyoshiro, and Waseshiro) among eight cultivars by inoculation to seedlings. External and internal symptoms of these isolates were not distinct in potato plants. The virulence of these isolates to potato was very low as compared with Verticillium dahliae. These two isolates were not pathogenic to Chinese cabbage, eggplant, green pepper, larkspur, parsley, snapdragon, soybean, tobacco, and tomato. This is the first report of V. tricorpus from potato in Japan.     

Differential effects of <Emphasis Type="Italic">Paenibacillus</Emphasis> spp. on cucumber mycorrhizas

The effects of 17 Paenibacillus strains on root colonization by Glomus intraradices or Glomus mosseae and plant growth parameters (shoot and root weight) of mycorrhizal cucumber plants were examined. The Paenibacillus strains were originally isolated from mycorrhizal (G. intraradices) and non-mycorrhizal cucumber rhizosphere and/or hyphosphere, except for strain EJP73, which originated from a Pinus sylvestris-Lactarius rufus ectomycorrhiza. Root colonization of cucumber plants by G. intraradices or G. mosseae was unaffected by all seven strains of Paenibacillus polymyxa, but was decreased or increased by four strains of Paenibacillus macerans and strain EJP73 of Paenibacillus sp. Overall, shoot dry weight of cucumber grown in symbioses with either G intraradices or G. mosseae was unaffected by inoculation with all of the Paenibacillus strains, except for strain MB02-429 of P. macerans, which increased the shoot dry weight in the cucumber-G. mosseae symbiosis. On the other hand, several Paenibacillus strains caused altered root growth. Three strains of P. polymyxa and four strains of P. macerans increased the root fresh weight of the cucumber–G. intraradices symbiosis, whereas three strains of P. polymyxa and one strain of P. macerans as well as Paenibacillus sp. EJP73, decreased the root fresh weight of the cucumber–G. mosseae symbiosis. In conclusion, our results show that bacteria from several species of Paenibacillus differentially affect cucumber mycorrhizas.     

Somatic embryogenesis,organogenesis and plant regeneration in taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> var. <Emphasis Type="Italic">esculenta</Emphasis>)

Callus was initiated in three different “esculenta” taro cultivars by culturing corm slices in the dark on half-strength MS medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by subculture of all corm slices to half-strength MS medium containing 1.0 mg/l thidiazuron (TDZ). Depending on the cultivar, 20–30% of corm slices produced compact, yellow, nodular callus on media containing TDZ. Histological studies revealed the presence of typical embryogenic cells which were small, isodiametric with dense cytoplasms. Somatic embryos formed when callus was transferred to hormone-free medium and ~72% of the embryos germinated into plantlets on this medium. Simultaneous formation of roots and shoots during germination, and the presence of shoot and root poles revealed by histology, confirmed that these structures were true somatic embryos. Plants derived from somatic embryos appeared phenotypically normal following 2 months growth in a glasshouse. This method is a significant advance on those previously reported for the esculenta cultivars of taro due to its efficiency and reproducibility.     

Effect of sucrose application,minerals, and irradiance on the <Emphasis Type="Italic">in vitro</Emphasis> growth of <Emphasis Type="Italic">Cistus incanus</Emphasis> seedlings and plantlets

To study the effect of sucrose on the sink-source relationship in in vitro-grown plants, Cistus incanus seedlings and plantlets were grown horizontally in a two-compartment Petri dish (split dish), with the root system in one compartment and the shoot in the other. Shoots and roots were exposed to different sucrose concentrations (0–30 g dm⁻³), two irradiance levels (25 and 160 μmol m⁻²s⁻¹) and the presence or absence of a minimum medium containing minerals and vitamins (M medium). Root and shoot biomass of the seedlings was enhanced by an increase in irradiance when the growth medium was not supplemented with sucrose indicating the role of photosynthesis in biomass production. When sucrose was added to either organ growth was enhanced as well. In the presence of sucrose in the root compartment, sucrose applied to the shoot compartment enhanced growth of both organs under low irradiance, while under high irradiance, sucrose had no further additive effect. In the absence of sucrose in the root compartment, the enhancement of root biomass by sucrose added to the shoot compartment was lower under high irradiance than under low irradiance. The response of Cistus plantlets to sucrose and irradiance differed from that of seedlings, probably reflecting a greater susceptibility of the plantlets to sucrose feedback inhibition on photosynthesis and biomass accumulation. The decrease in root and shoot growth when M medium was added to the shoot compartment and the relatively better growth of these organs when the roots were supplied with minerals and the shoot with sucrose, indicate that growth of the two organs in our experimental set-up was regulated by opposing fluxes of C and nutrients.     

Compatibility of <Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> as dual inoculants in black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

The compatibility of two biological inoculants, Trichoderma harzianum, a mycoparasitic biological control fungus and Piriformospora indica, a root colonizing plant-growth promoting endophytic fungus was evaluated using tissue cultured black pepper plantlets. We report, for the first time, the ability of P. indica to colonize black pepper, a perennial climber. T. harzianum inhibited the growth of P. indica in an in vitro dual culture plate assay. Simultaneous inoculation with both biological inoculants of tissue cultured black pepper plantlets negatively influenced root colonization by P. indica. However, when P. indica was applied initially followed 30 days later by T. harzianum, there was increased root colonization by the root endophyte P. indica and beneficial effects were found on the growth of the black pepper plants. The present study also showed that the efficacy of inoculation of the two fungal biological agents can be increased by sequential application of P. indica at the hardening stage followed by T. harzianum during transplanting into a soil-sand mixture.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated transformation of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> and production of flavonoids in hairy roots

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A₄GUS, R1000 LBA 9402 and ATCC11325. The A₄GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15–20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B₅ medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A₄GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1–30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.     

Isozymes variability among <Emphasis Type="Italic">Fusarium udum</Emphasis> resistant cultivars of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) (Millsp)

The present study was carried out to select the different pigeonpea cultivars for resistance against wilt caused by Fusarium udum and to assess the genetic variability among the resistant and susceptible cultivars. These cultivars were screened by root dip inoculation and classified into resistant (ICP 8863 and 9145), moderately resistant (ICP 11681 and Selection-1), susceptible (ICP 7118, TRG-1 and LRG-30) and highly susceptible cultivars (ICP-2376 and LRG-41). The peroxidase activity (PEO) in both leaf and root tissues of four pigeonpea cultivars (ICP 8863, Selection-1, ICP 2376 and LRG-30) were determined at 1^st, 4^th and 7^th day after inoculation (DAI) in healthy and F. udum infected tissues. Higher PEO activity in both leaf and root was observed and at 4^th DAI in susceptible cultivars. In native-PAGE analysis of isozymes, the induction of specific leaf peroxidase band (Em=0.17) and two root peroxidase bands (Em=0.24 and 0.55) were observed in ICP 8863 after inoculation. Significant differences were observed in the leaf phosphatase and esterase banding profiles of all the cultivars. The presence of leaf phosphatase band at Em of 0.04 was observed only in ICP 8863 and 11681. The leaf esterase band (Em=0.3) was well expressed in ICP 8863 when compared to other cultivars. The significance of peroxidase in plant defense mechanism and utility of biochemical markers in breeding programmes are discussed. Part of M.Sc. (Ag) thesis of the first author and approved by the Acharya N.G. Ranga Agricultural University during March 2002.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration in six cultivars of <Emphasis Type="Italic">Capsicum</Emphasis> spp. using different explants

In vitro regeneration from leaf, cotyledon and hypocotyl explants of six cultivars belonging to three species of Capsicum was achieved by direct organogenesis. The cultivar Umorok showed the best response while Meiteimorok, Haomorok, Mashingkha and Uchithi showed intermediate response and the cultivar Chiengpi was the least responsive. Leaf and cotyledon explants regenerated more shoots than hypocotyl explants and the maximum number of shoots were produced on Murashige and Skoog (1962) medium containing 8.8 μM 6-benzylaminopurine (BAP) with 11.4 μM indole-3-acetic acid (IAA). Elongation of shoot buds derived from different explants was achieved on medium containing 2.8 μM IAA and the elongated shoots were rooted on medium containing 2.8 or 5.7 μM IAA and 2.4 or 4.9 μM indole-3-butyric acid (IBA). Four-week old rooted plantlets were hardened and transplanted to the soil. The plantlets showed 90 % survival during transplantation.     

<Emphasis Type="Italic">In vitro</Emphasis> effects of GA<Subscript>3</Subscript> on morphogenesis of CIP potato explants and acclimatization of plantlets in field

Improvement of potato has been accomplished using conventional and non-conventional approaches coupled with numerous tissue culture procedures. The aim of the present study was to assess the efficacy of gibberellic acid (GA₃) on the morphogenesis of International Potato Center (CIP) potato explants and acclimatization of plantlets in the field. Nodal segments as an explant source (1–1.5 cm) were isolated from 31 CIP potato plantlets and were inoculated into Murashige and Skoog (MS) medium supplemented with 0.0 (control), 0.1, 0.5, or 1.0 mg L^?1of GA₃. The variation in growth parameters of the cultivars was then observed. The highest shoot induction occurred in MS medium containing 1.0 mg L^?1 GA₃ with an increase in the inter-nodal distance between nodes as compared to other treatments. Higher concentration (1.0 mg L^?1) of GA₃ significantly increased plant height and root length in the treated germplasm however; this concentration was inhibitory to the number of nodes and roots per plant. The number of leaves was significantly increased in plants receiving GA₃ treatment at lower concentration (0.1 mg L^?1). The 31 CIP genotypes were transplanted to the field and checked for yield quality traits. It was concluded from the results that GA₃ had significant effects on morphogenesis and was effective in the acclimatization of CIP potato plantlets in field.     

Effect of mutations in the pea genes <Emphasis Type="Italic">Sym33</Emphasis> and <Emphasis Type="Italic">Sym40</Emphasis>

Two pea (Pisum sativum L.) symbiotic mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread development and function in symbiotic root nodules have been characterised in terms of mycorrhizal colonisation of roots, shoot and root biomass accumulation and shoot and root phosphorus (P) content. The mutation in gene sym33 decreased mycorrhizal colonisation of roots (except arbuscule abundance in mycorrhizal root fragments, which increased) but did not change the effectiveness of mycorrhiza function. The mutation in sym40 did not affect either of these processes. Both mutants showed differences in plant development compared with the wild-type line SGE. The mutants had delayed flowering and pod ripening, and shoot/root biomass ratios and P accumulation also differed from those of SGE. These observations suggest that the gene mutations cause systemic changes in plant development.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation from young and mature explants of thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> and <Emphasis Type="Italic">T. longicaulis</Emphasis>) resulting in genetically stable shoots

An efficient in vitro propagation protocol, applicable both to young and mature explants of two Thymus spp., results in genetically stable plantlets. In vitro-grown shoot tips of Thymus vulgaris L. were exposed to cytokinins (6-benzyladenine, kinetin, and thidiazuron) alone or in combination with auxins, gibberellic acid (GA₃) and/or silver nitrate in order to optimize in vitro shoot proliferation. Optimum shoot proliferation (97% regeneration rate, with 8.6 shoots produced per explant) was obtained when semi-solid Murashige and Skoog (MS) medium was supplemented with 1 mg L⁻¹ kinetin and 0.3 mg L⁻¹ GA₃. Rooting of the shoots was easily obtained on semi-solid MS medium that was either hormone-free or supplemented with auxins. However, the best root apparatus (92.5% rooting rate, with 19 adventitious roots per shoot) developed on MS medium supplemented with 0.05 mg L⁻¹ 2,4-dichlorophenoxyacetic acid. Genetic stability was confirmed in the in vitro-germinated mother plant as well as the shoots that underwent two, four, six, eight, or ten cycles of in vitro subculturing by random amplified polymorphic DNA (RAPD) analysis. When applied to the micropropagation of mature shoot tips of T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas, the optimized in vitro propagation protocol resulted in a 97.5% shoot regeneration rate, with five shoots formed per explant, and 100% rooting. Rooted plantlets of both species were transferred to 250-mL plastic pots and successfully acclimatized by gradually reducing the relative humidity.