Morphogenesis and tissue cultures of three citrus species

Studies were performed to define tissue culture techniques and culture conditions for morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis (L.) Osb.), citron (C. medica L.) and lime (C. aurantifolia) (Christm. Swing). The optimal concentrations of NAA to induce root formation on stem segments were 10 mg l^-1 for sweet orange and lime, and 3 mg l^-1 for citron. The optimal BA concentration for shoot and bud proliferation was 3 mg l^-1 for sweet orange and citron, and 1 mg l^-1 for lime. Callus initiation was accomplished in a culture medium containing 10 mg l^-1 NAA and 0.25 mg l^-1 BA. Callus was maintained by periodical subculture into the same medium supplemented with 10% (v:v) organge juice. In vitro plantlets of the three species were obtained by rooting of shoots developed from bud cultures, and of citron and lime by development of shoots from root cultures. The plants were successfully established on soil.     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Callus formation and plantlet development from axillary buds of taro

Excised lateral buds of taro [Colocasia esculenta var. esculenta (L.) A.F. Hill] developed into plantlets and formed callus if cultured on media containing taro extract. α-Naphthaleneacetic acid enhanced the process but only if taro extract was also present. The tissue requirements for this variety of taro are different from those of Colocasia esculenta var. antiquorum (L.) A.F. Hill.     

Induction of adventitious buds in cultured petal explants ofChelidonium majus L.

Petal explants ofChelidonium majus L. (Papaveraceae) formed noteworthy adventitious buds without any intermediate callus when cultured under appropriate conditions. Bud formation was favored by combinations of 1–2 mg/l indoleacetic acid (IAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–0.5 mg/l kinetin (K). In the present study, neither bud formation nor callus formation occurred in cultures of excised leaves. A histological study revealed that adventitious bud formation occurred only in single epidermal layers of petals, while several subepidermal parenchyma layers did not join in its formation. Activation zones arising from the epidermis underwent intense cell divisions to initiate buds on the epidermal surface. These buds later turned green in color, developing into shoots which eventually grew into plantlets after root formation.     

Probable identity of substances in citrus that repress asexual embryogenesis

Summary The embryogenetic responseof culturedDaucus carota L. ‘Queen Anne's Lace’ callus was employed to attempt fractionation and identification of a repressive factor produced byCitrus medica L. ovules. The factor was evidently synthesized and released into the medium continuously, inasmuch as citron ovules that had been autoclaved with the medium were completely infeffective. The inhibition could be attributed to volatile and nonvolatile components. A substantial part of the inhibition was prevented by continuously refereshing the atmosphere within the cultures with filtered air. Monitoring of the gases produced by citron ovule sections under conditions simulating bioassays disclosed significant evolution of carbon dioxide, ethylene and ethanol. Repression of embryogenesis was not averted by trapping the liberated ethylene. On the other hand, ethanol in concentrations equivalent to those released by citron ovules suppressed asexual embryogenesis dramatically. The adverse effect of ethanol was reversed immeditaley upon transfer to ethanol-free medium. Another investigation had disclosed anti-embryogenetic effects of auxin, abscisic acid and gibberellin. Analysis ofCitrus ovules excised from young fuits disclosed those of monoembryonic citron to contain concentrations of IAA, ABA and GA₃ several times higher than those of polyembryonic Ponkan mandrain. The nonvolatile protion might be identified with these hormonal substances. This paper is part of B. Tisserat's PhD. dissertation in Botany at the University of California, Riverside. The research was supported in part by the Elvenia J. Slosson Fellowship in Ornamental Horticulture awarded to T. Murashige.     

The influence of citrus fruit condition on egg laying by the false codling moth, Cryptophlebia leucotreta

The oviposition response of Cryptophlebia leucotreta (Meyrick) (Lepidoptera: Tortricidae) to healthy navel and Valencia orange, Citrus sinensis (L.), fruit was measured in experimental orchards at Nelspruit, eastern Transvaal, South Africa, and was compared with the response to prematurely ripened fruit and injured fruit. Both premature ripening and injury increased the number of eggs laid on these fruit. Damaged fruit was almost twice as attractive as ripe fruit if the number of eggs laid can be used as a measure of attraction. Large wounds were no more stimulating than small lesions. The quantity of stimulus was not statistically significant, yet five or ten treated fruit in a cluster led to more egg-laying per fruit than a single treated fruit. More eggs were observed on healthy fruit within 50 cm of clusters of treated fruit than on healthy fruit on different trees, but this effect was not statistically significant.

---

Résumé La ponte de c. leucotreta Meyrick (Lep.; Tortricidae) sur oranges (Navel et Valencia) saines a été évaluée dans des vergers expérimentaux de Nelspruit à l'est du Transvaal, en Afrique du Sud. Elle a été comparée à la ponte observée sur des fruits prématurés ou endommagés. Ces deux dernières situations ont augmenté l'effectif d'ufs pondus sur les fruits; si le nombre d'ufs pondus est utilisé comme critère d'attraction, les fruits endommagés étaient presque deux fois plus attractifs que les autres. La taille des blessures est sans effet. Des fruits ont été artificiellement endommagés ou conduits prématurément à maturité avec un badigeon d'acide 2-chloréthylphos-phorique. Ces stimului n'ont pas eu d'effet cumulatif significatif; pourtant la ponte était plus importante sur des paquets de 5 ou 10 fruits artificiellement modifiés, que sur des fruits modifiés isolés. Une ponte plus importante a été observée sur des fruits sains à moins de 50 cm d'un groupe de fruits modifiés. Il est probable que des signaux visuels et olfactifs orientent la découverte du fruit par la tordeuse. Ces résultats suggèrent que les substances volatiles ont plus d'influence que la couleur ou la taille du fruit.La protection contre cet insecte devrait comprendre l'élimination des fruits mûris prématurément ou abîmés, mais il faudrait préciser si les fruits endommagés sont ou non découverts par hasard. La concentration des pontes sur et autour de ces fruits peut aussi réduire les pontes dans le reste du verger.

     

Growth responses and vascular plugging of citrus inoculated with rhizobacteria and xylem-resident bacteria

Summary Bacteria, isolated from roots (xylem tissue) of healthy and Young Tree Decline (YTD, Blight)-affected citrus trees, and also from nursery seedlings, were screened for potential pathogenicity by the tobacco hypersensitive reaction (HR). A majority (>75%) of the HR positive strains were classified as nonfluorescent pseudomonads. These HR positive strains were subsequently inoculated into rough lemon (Citrus jambhiri Lush.) and sweet orange (C. sinsensis Osbeck) seedlings or into Valencia sweet orange budded on rough lemon root-stock. Many of the HR positive pseudomonads reduced fresh weights (up to 94%) of roots and shoots and some reduced xylem water conductance and caused scion dieback. There was no evidence of necrosis or root rot in inoculated roots. A few HR negative Pseudomonas and Enterobacter strains significantly, but less severely, inhibited (to 43%) root growth of sweet orange seedlings. HR negative mutants derived from HR positive strains were considerably less inhibitory. Postinoculation stresses (dark and cold) markedly decreased susceptibility of seedlings to bacterial-induced inhibition. Evidence of cultivar-specific effects was obtained in comparable inoculations of rough lemon and sweet orange seedlings. Soil application of a fluorescent pseudomonad, which alone was growth stimulatory, intensified inhibitory effects of nonfluorescent, growth inhibitory, psuedomonads. This study demonstrates that many rhizobacteria isolated from xylem tissue of roots have detrimental effects on citrus.     

Influence of carbon source and concentration on the in vitro development of olive zygotic embryos and explants raised from them

The influence of sucrose or mannitol on in vitro zygotic embryo germination, seedling development and explant propagation of olive tree (Olea europaea L.) was compared. Embryos germinated without sucrose in the medium but for adequate development of the seedlings to yield viable plants, a carbohydrate supply was necessary; both sucrose and mannitol were equally suitable for this purpose. However, when explants obtained from in vitro germinated embryos were cultured with mannitol or sucrose, then the polyalcohol promoted significantly more growth than sucrose by increasing shoot length, pairs of leaves formed, and breaking apical dominance. This improved the in vitro culture of olive plant material, thus allowing new olive clonal lines to be obtained in shorter times. This will assist in future breeding experiments with the species.     

Repression of asexual embryogenesis in vitro by some plant growth regulators

Summary A factor that represses asexual embryogenesis has been observed in the Rutaceae, with particularly high concentrations in the naturally monoembryonic cultivars. This investigation was an initial step towards identifying the factor.Citrus reticulata Blanco Ponkan mandarin nucellus explants andDaucus carota L. ‘Queen Anne's Lace’ callus were employed to examine effects of known plant growth regulators and to determine possible identity of one or more of them with the repressive factor. The chalazal halves of ovules ofC. media L. ‘Citron of Commerce’ were used as control repressor source. Embryo initiation and growth of both test tissues were depressed markedly by 2,4-D, abscisic acid and ethephon. Slight inhibitions were obtained with IAA, kinetin and gibberellic acid. Recovery from the repressor did not occur readily inCitrus nucellus following recultures in citron-ovule-free medium; carrot callus resumed normal embryogenesis immediately upon transfer to suppressor-free medium. The repression by natural sources apparently involved the combined action of some or all natural hormones that are generically related to the above. This paper is part of B. Tisserat's Ph.D. dissertation in Botany at the University of California, Riverside. The research was supported in part by the Elvenia J. Slosson Fellowship in Ornamental Horticulture awarded to T. Murashige.     

Distribution ofGeotrichum candidum citrus race in citrus groves and non-citrus fields in Japan

The presence ofGeotrichum candidum citrus race, the citrus sour rot pathogen, was examined in soils of citrus groves and non-citrus fields of Japan. Soil samples were collected from 223 sites (118 sites in citrus groves, and 105 sites in fields cultivated with 33 species of non-citrus plants and in evergreen broad-leaved forest) in 11 main citrus-growing prefectures, and Hokkaido, a non-citrus-growing area. Of 236 soil samples from citrus groves, 95.76% containedG. candidum citrus race and 0.42% contained the non-citrus race; while of 210 samples from non-citrus fields, 62.85% and 4.76% contained the citrus race and the non-citrus race respectively. All of the citrus race isolates obtained either from citrus groves or non-citrus fields were pathogenic on lemon (Citrus limon) and satsuma mandarin (Citrus unshiu), but some of these isolates failed to infect orange (Citrus sinensis). The non-citrus races were pathogenic on ripe tomato fruit (Lycopersicon esculentum) and ripe muskmelon fruit (Cucumis melo var.reticulatus). Results indicated that citrus sour rot pathogen is widely distributed in citrus groves and non-citrus fields of diverse plant species in Japan.     

Production and use of calli from yellow birch buds for in vitro assessment of virulence of Nectria galligena isolates

Field studies of perennial Nectria canker of northern hardwoods, caused by the ascomycete fungus Nectria galligena, are time-consuming since the disease develops slowly on the stem of trees. In this report, an in vitro bioassay is described for determining and comparing the virulence of different isolates of Nectria galligena by using calli produced from yellow birch buds. The technique facilitates the distinction between highly virulent and less virulent isolates of the pathogen within one week following the inoculation.     

Genotypic response of cowpea Vigna unguiculata (L.) to in vitro regeneration from cotyledon explants

Summary A plant regeneration system applicable to 17 cowpea genotypes was developed. Cotyledons were initiated on 1/3 MS medium containing 15 to 35 mg N⁶-benzyladenine (BA) per 1 (66.6 to 155.3 μM) for 5 to 15 d. For shoot regeneration, the explants were transferred to a medium containing 1 mg BA per 1 (4.4 μM). Within 1 wk, shoot formation was visible at the proximal end of the cotyledons. Regeneration percentages (1% to 11%) and the numbers of shoots (4 to 12 per explant) were significantly influenced by genotype. Culture duration and BA concentration in the initiation stage significantly affected regeneration capacity. Explants initiated on media containing 15 mg BA per 1 for 5 d resulted in the highest percentage of explants capable of regeneration. Conversely, the highest number of shoots was obtained from explants initiated on media supplemented with 35 mg BA per 1. Whole plants were obtained on a plant growth regulator-free medium. To our knowledge, this is the first report of plant regeneration from U.S. commercial cowpea cultivars and breeding lines. This system is adaptable to diverse cowpea genotypes and will facilitate cowpea genetic transformation. Published with the approval of the Director of the Arkansas Agricultural Experiment Station.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Development of shoot inHydrangea macrophylla I. Terminal and axillary buds

The structure of shoots, in particular of winter buds, ofHydrangea macrophylla was examined. The non-flower-bearing shoot is usually composed of a lower and an upper part, between which a boundary is discernible by means of a distinctly short internode. This internode is the lowermost of the upper part, and it is usually shorter than the internodes immediately above and below, although the internodes tend to shorten successively from the proximal to the distal part of the shoot. Variations exist in the following characters among the terminal bud, the axillary bud on the lower part of the shoot and the axillary bud on the upper part: (1) length of bud; (2) character of the outermost pair of leaf primordia; (3) degree of development of secondary buds in the winter bud; and (4) the number of leaf primordia. Usually, the terminal bud contains several pairs of foliage leaf primordia with a primordial inflorescence at the terminal of the bud, but the axiallary bud contains only the primordia of foliage leaves in addition to a pair of bud scales.     

Selection and breeding for salinity tolerancein vitro

Summary Selection for tolerance to NaCl inCitrus sinensis andC. aurantium has been carried out in agar and suspension cultures. Callus was subjected to culture media containing up to 0.17M NaCl for ten passages. Selected cell lines were grown for three passages on media without salt before further tests on saline media. Four stable tolerant cell lines, differing in degree of tolerance, have been selected fromC. sinensis. Four lines of similar tolerance have been selected fromC. aurantium. The stability of most lines was very satisfactory. MostC. sinensis lines grew well in media containing up to 0.2M NaCl, andC. aurantium lines in media of up to 0.15M NaCl.Embryos were regenerated in most selected cell lines fromC. sinensis and, more sporadically, fromC. aurantium. Addition of 0.5–0.6% NaCl to the media often enhanced embryogenesis. Embryos from a selected line ofC. sinensis showed higher tolerance to NaCl in the medium than comparable embryos from an unselected line.Single embryos derived from both selected and unselected cell lines ofC. sinensis were successfully cloned. A limited comparison of plantlets from one tolerant line (R14) with plantlets from unselected control lines showed better adaptation of the former to salt (0.085 to 0.12M NaCl in the medium), and a lesser degree of leaf burn symptoms.Contribution No. 1045-E, 1984 series.     

Endogenous IAA levels and development of coffee flower buds from dormancy to anthesis

Dormant coffee (Coffea arabica L.) flower buds require water stress to stimulate regrowth. A xylem specific water-soluble dye, azosulfamide, was used to quantify water uptake of buds after their release from dormancy by water stress. In non-stressed flower buds, the rate of water uptake was generally slower and variable compared to stressed flower buds, where the rate of uptake tripled from 1 to 3 days after rewatering and preceded the doubling of fresh and dry weight of buds. Free, ester and amide IAA levels of developing flower buds were measured by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution technique with [¹³C₆]IAA as an internal standard. Throughout development, the majority of IAA was present as amide IAA. The proportions of amide and free IAA increased one day after plants were released from water stress, and preceded the doubling of fresh and dry weight. Free and conjugated IAA content per bud remained stable during the period of rapid flower growth until one day before anthesis.Abbreviations FW fresh weight - IAA indole 3-acetic acid - HPLC high performance liquid chromatography - GC-MS-SIM gas chromatography-mass spectrometry selected ion monitoring - NAA naphthalene acetic acid - IBA indole butyric acid     

Recovery of whole plants of sweet orange from somatic embryos subjected to freezing thawing treatments

Freezing/thawing conditions for cryopreservation of somatic embryos of Washington Navel sweet orange (Citrus sinensis (L.) Osb.) were evaluated. No survival of fast-cooled embryos occurred regardless of the thawing method. Embryos subjected to slow cooling at an estimated rate of 0.5°C min^-1 down to –42°C followed by immersion in liquid nitrogen survived. Survival rate depended on the thawing method. An average survival of 30.5% was achieved when frozen embryos were thawed by immersion in a water bath at 37°C. Surviving embryos developed into whole plantlets and no phenotypic abnormalities have been observed during a growth period of four years. Total soluble proteins and peroxidase and esterase isoenzyme analysis did not show differences between treated plants and non-frozen controls.     

Shoot regeneration from petioles of coral bells (Heuchera sanguinea Engelm.) cultured in vitro, and subsequent planting and flowering ex vitro

Summary Successful shoot regeneration from petioles, leaves, and petioles with leaves cultured in vitro is reported in Heuchera sanguinea. Petioles or petioles with leaves regenerated more shoots than leaves alone. For culture, the optimum hormonal concentrations were 0.19 μM α-naphthaleneacetic acid combined with 0.44 or 4.4 μM benzyladenine in Murashige and Skoog-based (MS) medium: the regenerating rate and the number of shoots per explant were 60% and 8.6–9.7, respectively. Histological study on petiole culture showed dividing cell clusters including vascular tissues after 1 wk, callus including several dividing cell clusters at the periphery after 3 wk and then apical meristems with immature leaves after 5 wk. Rooting from the regenerated shoots was highest (95%) on MS medium containing 4.9 μM indole-3-butyric acid. Seventy-three percent of rooted plants were successfully acclimatized in pots. When they were cultured in the field, the plants grew and most flowered the following year over winter.     

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.     

Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

Summary A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL^–1 and NAA concentration of 1mgL^–1. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium — mediated transformation protocols involving cotyledonary petioles.