Gibberellin enhancement of the enzymic release of Pisum root cell protoplasts

Cultivar differences have been reported in the protoplast yields from Pisum sativum root cortical explants treated with preparations of commercial cellulase and pectinase. The presence of intracellular starch significantly influenced these protoplast yields. The application of gibberellin before or during the enzymic wall-degradation increased the protoplast yields from two of the five cultivars tested. For tissues of'Little Marvel' pea roots, 10 mg 1⁻¹ of gibberellin most effectively increased the release of protoplasts when the hormone preceded the enzyme incubation. One mg 1⁻¹ of gibberellin was most effective at increasing the protoplast release when the tissues were treated with the hormone simultaneously with the wall-degrading enzymes. Mitotic activity was significantly reduced in protoplasts derived from gibberellin-treated tissues.     

In vitro Organogenesis in Explants from Different Cultivars of Begonia X hiemalis

Petiole explants from 17 cultivars of Begonia X hiemalis were grown on a basal agar medium with different combinations of NAA and BA as well as on media lacking microelements or vitamins. The stock plants were kept either under short days (7–8 h of light perday) at 15°C or under long days (15–16 h of light) at 18–21°C. The day length during the in vitro culture was 20 h of light and the temperature 21°C. Explants from short-day treated stock plants did not show any differentiation. In explants from long-day treated stock plants, the percentage of explants with shoot, root and with both shoot and root initiation were recorded after 55 days. Explants forming both shoots and roots were transferred to soil, and plantlet formation was observed after another 55 days. The percentage of explants with organ and plantlet formation differed between cultivars. With increasing NAA and decreasing BA concentrations, the percentage of explants forming only roots increased, whereas the percentage of explants with only shoots decreased. Plantlet formation was most frequent in explants from NAA: BA ratios of 2: 1 and 10: 1, and a variation was found between different cultivars. When the vitamin fraction was not added to the medium, this did not influence formation of shoots, roots and plantlets. When the microelements were omitted. shoots, roots and callus were formed, but no plantlets.     

Direct fermentation of various pure and crude starchy substrates to L(+) lactic acid using Lactobacillus amylophilus GV6

Lactobacillus amylophilus GV6 fermented a variety of pure and natural starches directly to L(+) lactic acid. Starch to lactic acid conversion efficiency was more than 90% by strain GV6 at low substrate concentrations with all starches. The strain GV6 produced high yields of lactic acid per g of substrate utilized with pure starches such as soluble starch, corn starch, and potato starch, yielding 92–96% at low substrate concentrations in 2 days and 78–89% at high substrate (10%) concentrations in 4–6 days. Strain GV6 also produced high yields of lactic acid per g of substrate utilized with crude starchy substrates such as wheat flour, sorghum flour, cassava flour, rice flour and barley flour yielding 90–93% at low substrate concentrations in 2 days and 80% or more at high substrate concentrations in 6–7 days. Lactic acid yields by L. amylophilus GV6 with pure starches were comparable when low cost crude starchy substrates were used. Lactic acid productivity by strain GV6 is higher than for any other previously reported strains of L. amylophilus.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

Transformation of potato using mannopine and cucumopine strains of Agrobacterium rhizogenes

Mannopine and cucumopine strains of Agrobacterium rhizogenes were used for genetic transformation in two cultivars of potato (Solanum tuberosum L.). An overnight pretreatment of stem fragments with NAA prior to bacterial infection was necessary to induce root formation, otherwise very few roots were produced. Whatever the potato cultivar used, rhizogenesis induced by NAA pretreament depended on the bacterial strain. In fact, when explants from both potato cultivars were pretreated with 26.5 M NAA, on average 84.4% and 71.9% produced roots after inoculation with the strains 2659 and 2659 GUS respectively. On the contrary, few rhizogenic responses (2.0–17.0%) or no response at all (0.0%) were obtained with the strains 15834 and 8196 GUS whatever NAA concentration used. Tests for confirming stable transformation of plant explants by examining both -glucuronidase activity and the presence of opines showed that 85% of the selected roots were cotransformed. Most of the transformed roots were highly branched and grew rapidly, compared to non-transformed roots with no branching and poor growth. Transgenic plants were readily regenerated with a frequency reaching 80% of total explants tested for both potato cultivars.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - df degree of freedom - F F distribution - GA₃ gibberellic acid - MS Murashige and Skoog basal medium - NAA -naphthaleneacetic acid - P probability - T-DNA transferred DNA     

Comparative host tissue Réactions of Musa acuminate (AAA group) cvs Poyo and Gros Michel roots to three banana-parasitic nématodes

In a comparative histopathological investigation, Poyo and Gros Michel cul-tivars of Musa acuminata (AAA triploid) were inoculated with Radopholus similis, Helicotylenchus multicinctus or Hoplolaimus pararobustus and were grown in a greenhouse under tropical conditions. R. similis infected all the cortical parenchyma layers of the roots, reaching the vascular cylinder, but it stayed more superficial in Gros Michel roots. Red-brown cytoplasmic globules appeared in the cortical parenchyma cells of Gros Michel only. H. multicinctus infected much of the outer cortical parenchyma in roots of both cultivars with a few phenolic cells occurring around the superficial lesions. H. pararobustus penetrated only the immediate sub-epidermal tissues in both cultivars. The differences observed between nématodes and cultivars reflect specific host-nematode interactions on bananas.     

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.     

In vitro response of strawberry cultivars and regenerants to Colletotrichum acutatum

Diseases affecting strawberry (Fragaria × ananassa Duch.) have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose, caused by the fungus Colletotrichum acutatum, is one of the most serious diseases of strawberry worldwide. Tissue-culture induced (somaclonal) variation provides one strategy for generating disease-resistant genotypes. As part of a program to generate strawberry germplasm resistant to anthracnose, an in vitro screening system was used to evaluate several commercial cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie propagated in vitro, and shoots regenerated from leaf explants of these cultivars for resistance to C.␣acutatum isolate Goff (highly virulent). Regenerants with increased levels of resistance were identified from all of the cultivars. The greatest increases in disease resistance were observed for regenerants from leaf explants of cultivars Pelican and Chandler that exhibited 17.5- and 6.2-fold increases in resistance, respectively. The highest levels of anthracnose resistance (2 to 6% leaf necrosis) were exhibited by regenerants from explants of cultivars Pelican and Sweet Charlie. These studies suggest that generating somaclonal variation may be a viable approach to obtaining strawberry plants with increased levels of anthracnose resistance.     

In vitro root differentiation and essential-oil accumulation in Angelica archangelica

Summary We studied the relationship between root differentiation and the accumulation of essential oils in Angelica archangelica in in vitro cultures and in the intact plant. Root regeneration was obtained using stem and leaf explants subjected to treatment with the auxins indole-3-butyric acid, indole-3-acetic acid and α-naphthaleneacetic acid. In both stem and leaf explants, treatment with indole-3-butyric acid induced the highest rhizogenic response in terms of both percentage of explants with roots and number of roots per explant. Independently of hormonal treatment, stem explants produced a higher average number of roots per explant. Root meristemoids were already visible at day 7 of culture in the treatments with indole-3-butyric acid and indole-3-acetic acid; they were formed directly by cambial-cell division. In vitro-regenerated roots retained primary root structure and differentiated only two primary ephemeral ducts in the pericycle; no accumulation of essential oils was detected. Same-size roots taken from the intact plant showed secondary structure and essential-oil accumulation. The results of this study suggest that the synthesis and accumulation of essential oils in Angelica archangelica is closely linked to the differentiation of secondary secretory ducts.     

Water use and chemical composition of ryegrass (Lolium) cultivars

Summary Significant differences in total dry matter yields of shoots and roots were found between 11 ryegrass (Lolium) cultivars grown in a glasshouse. Although shoot yield varied significantly between individual cultivars there was no overall difference between the annual and perennial cultivars; whereas for roots, the yields of the perennial plants were much smaller than those of the annual types. Water use (g H₂O g total DM^–1) also varied significantly between cultivars. However, there was no relationship between efficient water use and dry matter production.No significant differences were found in shoot composition between the cultviars for nitrogen, phosphorus, and potassium; however, concentrations of sulphur, magnesium, calcium, and sodium varied significantly. Sodium concentrations were generally higher in the annual compared to the perennial cultivars. For roots only nitrogen, phosphorus, and sulphur differed significantly between cultivars. Of the elements only calcium in the shoots was shown to be related to water use. Thus cultivars which were low users of water also had significantly lower calcium concentrations in their shoots. Water use appeared to affect the absorption of calcium by the root to a far greater extent than the transport from roots to shoot. An apparent relationship between magnesium concentration in the shoots and water use was shown to be due to the close association of magnesium with calcium in the plant.     

Energetics of nectar production in some strawberry cultivars as a predictor of floral choice by honeybees

Nectar productivity of thirteen cultivars of strawberry (Fragariq sp.) was examined in relation to the foraging activity ofApis cerana indica F. andApis mellifera L. The cultivars were found to vary in the quantity of nectar produced (0·020 to 0·735 ul/flower/day), nectar-sugar concentration (30 to 42%) and amount of sugar (0.006 to 0·2898 mg/flower/day). Consequently energy reward varied from 0·1037 to 4·851 joules/ flower/day. Foraging of bothApis cerana indicq andApis melliferq correlated with energy yields. The results suggest that cultivars with higher caloric rewards had a competitive edge over the others in attracting foraging populations of both species of the bee.     

The effects of anther culture and plant genetic background on Agrobacterium rhizogenes-mediated transformation of commercial cultivars and derived doubled-haploid Brassica oleracea

The production of transgenic roots was scored for eight Brassica oleracea cultivars from broccoli, cabbage, cauliflower and kale following inoculation with an Agrobacterium rhizogenes cell line carrying a binary plasmid bearing the green fluorescence protein (gfp) gene in the T-DNA. Significant differences in the numbers of explants producing transgenic roots were observed between cultivars, ranging from 1.4% for Marathon F1 to 57.8% for the Green Duke F1. Three F1 cultivars were subjected to anther culture, and doubled-haploid (DH) lines were used for transformation. The DH lines produced showed considerable variation for transgenic root production with some lines showing increased efficiency compared to the parental F1 cultivar. Grouping of the DH lines into response classes with respect to transgenic root production allowed the development of potential genetic models to explain the variation in performance released from each F1 cultivar. No apparent segregation distortion for transgenic root production was observed in the DH lines following anther culture.     

Metabolic profiles of six African cultivars of cassava (Manihot esculenta Crantz) highlight bottlenecks of root yield

Cassava is an important staple crop in sub‐Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenolpyruvate carboxylase activity support a C₃ mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin–Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse‐grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.     

Evaluation of the effect of <Emphasis Type="Italic">in vitro</Emphasis> stress and competition on tissue culture response of flax

This study was carried out to investigate the in vitro competition in tissue culture of three flax (Linum usitatissimum L.) cultivars using different distances among hypocotyl explants cultured. Hypocotyl fresh and dry masses, shoot regeneration percentage, shoot number per hypocotyl, regenerated shoot length and total chlorophyll content were examined during shoot regeneration, while plantlet height, number of roots and length of roots were recorded during rooting. With decreasing distance among explants we observed increased shoot regeneration and rooting till a certain point from where stress initiated and significant decreases in all parameters observed. Explants cultured at distance 1.0 cm were found to be at their optimum.     

Carbohydrate and Proline Contents in Leaves, Roots, and Apices of Salt-Tolerant and Salt-Sensitive Wheat Cultivars1

Intra-specific variations in nonstructural carbohydrates and free proline were determined in leaves, apices, roots, and maturing seeds of two salt-tolerant cultivars (CR and Kharchia-65) and one salt-sensitive cv. Ghods of spring wheat (Triticum aestivum L.) grown in sand culture at various levels of salinity (0, 100, 200, and 300 mM NaCl and CaCl₂ at 5 : 1 molar ratio) under controlled environmental conditions. The levels of leaf, apex, and root ethanol-soluble carbohydrates, fructans, starch, and proline increased in line with elevating level of salinity in all three cultivars under investigation. The contents of proline, soluble and insoluble carbohydrates in the apex increased to levels exceeding those in the leaves and roots. Soluble carbohydrate content of salt-sensitive cv. Ghods was higher in the leaves, apices, and roots and lower in the maturing seeds than in the other cultivars at all levels of salinity except at 300 mM. The results show considerable variation in the amount of soluble, insoluble sugars, and proline among plant tissues and wheat genotypes in response to salinity. Higher soluble carbohydrates and fructan in leaves, roots and maturing seeds of stressed plants indicate that their accumulation may help plant to tolerate salinity. Salt-sensitive cv. Ghods accumulated less soluble sugars in the maturing seeds and higher soluble sugars in the apices, which might be used as an indicator in screening wheat genotypes for salinity tolerance.     

Differential organ infection studies, potyvirus elimination, and field performance of virus-free garlic plants produced by tissue culture

Presence of potyvirus in single garlic (Allium sativum L.) cloves from the same bulb, and in five single leaves excised from commercial field-grown individual plants was studied using ELISA. It was found that the viruses were not present in all organs of the same plant, since some cloves of the same bulb were infected with potyvirus but some others were potyvirus-free. Analyzed leaves from a given plant also exhibited irregular distribution of potyvirus. This study also aimed to obtain potyvirus-free plants from two commercial garlic cultivars (Taiwan and Chileno) using cloves subjected to thermotherapy, chemotherapy or meristematic dissection followed by in vitro culture. Thermotherapy (sequential treatment at 32°C for a week, 36°C for 2 weeks, and 38°C for 3 weeks) was found to affect survival of explants and 36.5% cloves from Taiwan and 26.8% from Chileno cultivars were recovered after the treatment. ELISA tests showed that 63% of the cloves of Taiwan that survived the treatment and 70.9% of Chileno explants were potyvirus-negative. Regarding chemotherapy (205 μM Ribavirin solution), the explants (cloves) survived, but only an average of 27.0–34.8% were negative for the presence of potyvirus. When meristematic dissection was applied, an average of 41.7% explants of Taiwan and 34.2% of Chileno survived the treatment, and approximately 64% of these explants from both cultivars were potyvirus-negative. Potyvirus-free garlic plants grown in field conditions showed longer stems with a major fresh and dry weight per bulb, and also exhibited a higher yield than non-treated plants.     

High frequency plant regeneration from protoplasts in cotton <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM₈P medium with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 μM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 μM indole-3-butyric acid (IBA) and 0.93 μM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.     

Response of cassava to water stress

Summary Cassava (Manihot esculenta Crantz) is a staple food for a large sector of human population in the tropics. It is widely produced for its starchy roots by small farmers over a range of environments on poor infertile soils with virtually no inputs. It is highly productive under favorable conditions and produces reasonably well under adverse conditions where other crops fail. The crop, once established, cansurvive for several months without rain. There is a wide variation within the cassava germplasm for tolerance to prolonged drought and the possibility to breed and select for stable and relative high yields under favorable and adverse conditions does indeed exist. Research with several cassava clones at CIAT has shown that high root yield under mid—term stress is not incompatible with high yield under nonstress conditions. Plant types with high yield potential under both conditions (e.g. the hybrid CM 507-37) are characterized by having slightly higher than optimum leaf area index under nonstress conditions, higher leaf area ratio and more intensive and extensive fine root system.     

In vitro regeneration from hypocotyl and seedling cotyledons of tronchuda (Brassica oleracea var. tronchuda Bailey)

Four tronchuda (Brassica oleracea var. tronchuda Bailey) cultivars were tested for their ability to regenerate in vitro on Murashige & Skoog (MS) medium supplemented with 3 different combinations of -naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Explants were either axillary bud-free whole cotyledons or hypocotyls from 7-day-old darkgrown seedlings. The ability to regenerate varied by cultivars, explants and the concentration of growth regulators. Hypocotyl explants of all 4 cultivars, and cotyledon explants of 2 cultivars, developed plantlets within 4 weeks. Hypocotyl explants produced more shoots than cotyledons. Cotyledon explants produced more roots than hypocotyls. Best shoot regeneration was on MS medium supplemented with 2 mgl^-1 BAP and 0.1 mgl^-1 NAA. Portuguesa produced the most shoots. Some regenerants varied in leaf shape and phyllotaxy.Abbreviations BAP benzylaminopurine - NAA napththaleneacetic acid - IBA indolebutyric acid     

The evaluation of rhizomania resistant sugar beet for the UK

Sugar beet rhizomania disease, caused by Beet necrotic yellow vein virus and transmitted by the soil‐borne parasite Polymyxa betae, was first recorded in the UK in 1987. Recently, breeding lines and cultivars with partial resistance to the virus derived from the ‘Holly’ source of resistance have been developed and their suitability for use under UK conditions is explored in this paper. Virus multiplication in the roots of resistant lines exposed to severe disease pressure in glasshouse tests, when quantified by ELISA, was less than one third of that in susceptible controls. More recently developed resistant lines had a lower virus content, on average, largely due to a reduced frequency of susceptible individuals. There was no evidence for resistance to the vector, P. betae, in virus resistant lines. However, the proportion of viruliferous P. betae resting spores in the roots, estimated using the most probable number (MPN) technique, was reduced by at least one third in resistant lines compared with the most susceptible control. A novel line, containing an additional gene to that in ‘Holly’, was the most effective, reducing the infection level to 3% of that in the susceptible control. In two field experiments on severely infested sites, the rate of infection of a resistant line, when assessed by ELISA, was reduced by half compared with a susceptible cultivar and sugar yields of resistant lines were consistently 2–3 times higher than those of susceptible cultivars. In 41 trials on rhizomania‐free sites, several recently introduced resistant lines exhibited sugar yields and agronomic performance comparable to that of three selected high yielding, susceptible cultivars. Results are discussed in relation to the specific UK requirements for rhizomania resistant cultivars. One resistant line, Beta 805 (cv. Concept), fulfilled the requirements for widespread use to control the disease.