Influence of Boron on Somatic Embryogenesis in Papaya

Influence of boron on somatic embryogenesis in papaya (Carica papaya L.) cv. Honey Dew was investigated. Immature zygotic embryos were grown in the induction medium containing Murashige and Skoog basal salts, with B₅ vitamins, picloram (1 mg dm⁻³) or 2,4-dichlorophenoxy acetic acid (2 mg dm⁻³) and different concentrations of boric acid (30 to 500 mg dm⁻³). Maximum somatic embryo initiation was observed at 62 mg dm⁻³ boric acid irrespective of the growth regulator used. The cotyledonary stage somatic embryos were germinated on MS basal medium devoid of growth regulators. The regenerated plantlets were hardened under greenhouse conditions and transferred to field. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Culture of Immature Pea Embryos

Pea embryos of a range of developmental stages were culturedin a defined medium in vitro for up to 16 days. The criticalfactor for successful culture was the osmotic pressure of themedium; for the stages studied this was provided by the incorporationof 18 per cent sucrose in the medium. The growth of embryosof a range of genotypes was compared; small seeded genotypescould grow at comparable rates in vitro to those attained invivo. The amount of protein synthesized in vitro was similarto that attained in vivo, whereas slightly higher and lowerlevels of starch and DNA respectively were attained in vitro.The roles of intrinsic and extrinsic factors in regulating embryogrowth were studied by comparing the growth in culture of embryosof different genotypes and of hybrid embryos derived from reciprocalcrosses. embryo culture, pea embryos, hybrid embryos, osmotic pressure     

The In Vitro Culture of Immature Barley Embryos on Different Culture Media

Immature barley embryos (Hordeum distichum var. Julia) of between0•20 and 0•80 mm in length, were isolated from thedeveloping grain and cultured in vitro on various culture media.The subsequent development of the embryos was followed overa period of weeks, and where germination ensued the growth rateof shoot and root meristems was compared with in vivo germinationrates. Various growth media were assessed for their abilityto support normal development of immature embryos. A numberof published media failed to support satisfactory developmentof young embryos. The addition of 1–15 per cent coconutmilk to Norstog's Medium I (mineral + vitamin solns) enhancedembryo development and lowered the threshold of viability fromembryos of 0•50 mm in length to 0•35 mm. Althoughin many cases germination ensued, embryo development was largelyabnormal. A slightly greater enhancement of growth was achievedwith 0•05–0•30 per cent casein hydrolysate asthe growth medium supplement, although abnormal developmentwas not eliminated. A further lowering of the viability thresholdto include embryos of 0•25 mm in length was obtained bycombining 2•7 mM glutamine with the casein hydrolysatesupplement. Normal development and germination of embryos assmall as O25 mm was however obtained on Norstog's Medium JJand the results were reproduced in four additional if . distichumvarieties. In each case the critical threshold of viabilitywas found to lie in embryos of 0•20–0•30 mmin length.     

Morphological analyses and variation in carbohydrate content during the maturation of somatic embryos of <Emphasis Type="Italic">Carica papaya</Emphasis>

Efficient protocols for somatic embryogenesis of papaya (Carica papaya L.) have great potential for selecting elite hybrid genotypes. Addition of polyethylene glycol (PEG), a nonplasmolyzing osmotic agent, to a maturation medium increases the production of somatic embryos in C. papaya. To study the effects of PEG on somatic embryogenesis of C. papaya, we analyzed somatic embryo development and carbohydrate profile changes during maturation treatments with PEG (6%) or without PEG (control). PEG treatment (6%) increased the number of normal mature somatic embryos followed by somatic plantlet production. In both control and PEG treatments, pro-embryogenic differentiation to the cotyledonary stage was observed and was significantly higher with PEG treatment. Histomorphological analysis of embryonic cultures with PEG revealed meristematic centers containing small isodiametric cells with dense cytoplasm and evident nuclei. Concomitant with the increase in the differentiation of somatic embryos in PEG cultures, there was an increase in the endogenous content of sucrose and starch, which appears to be related to a rising demand for energy, a key point in the conversion of C. papaya somatic embryos. The endogenous carbohydrate profile may be a valuable parameter for developing optimized protocols for the maturation of somatic embryos in papaya.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

In vitro Culture of Abscissed Immature Avocado Embryos

The efficiency of an avocado hybridization programme, usingsmall potted glasshouse plants, was reduced by a high rate ofabscission of immature fruitlets bearing embryos too young forconventional germination. This was overcome in part by culturein vitro of the shed embryos on a liquid medium supplementedwith 0.5 mg l^–1 benzyladenine. Most embryos younger than6 weeks did not survive in culture, but older embryos slowlyproduced multiple shoots, with axillary shoot growth being furtherstimulated by removal of both cotyledons. Embryo response wasnot related to cultivar. Shoots removed from culture could begrafted to seedling rootstocks. Grafting was considered morereliable than dependence on growth of the main root or adventitiousroots in vitro to produce established plants. Persea americana Miller, avocado, abscissed fruitlets, in vitro embryo culture     

Embryo Rescue in Wide Crosses in Arachis. 2. Embryo Development in Cultured Peg Tips of Arachis hypogaea

Embryo rescue techniques in Arachis are potentially importantfor recovering interspecific hybrids which have the propensityto abort. Pegs are commonly produced in interspecific crosses,but either they fail to reach the soil because growth is arrested,or pods are produced but embryo development is never re-initiated.Peg tips, with the ovule and embryo, of A. hypogaea L. cv. ‘NC6’, were used to determine whether peg tips can be usedas nurse tissue for in vitro culture of embryos. Tissues werecollected 1, 2, 3 and 4 d after self-pollination, after whichpeg meristems were removed from half the pegs, and culturedon five media combinations. Continued reproductive developmentwas observed for embryos cultured at all four collection days;however, the highest frequency of growth was observed in 1-d-oldtissues. Evidence is presented that meristematic activity mayrestrict embryo growth in the 2- to 4-d-old embryos and, oncethe sequence of events is initiated to slow embryo growth, itis not easily reversed in vitro. Achievements of embryo growthto multicellular, globular stages (stages 1–1 or 1–2)encourage the development of methods to recover very young embryosthrough tissue-culture techniques. Embryo culture, morphology, interspecific hybridization, Arachis hypogaea, comparative light and scanning electron microscopy, peanuts, groundnuts     

The Use of Single Somatic Embryo Culture in Propagating and Regenerating Lucerne (Medicago sativa L.)

Embryogenic cultures of lucerne (Medicago sativa L.) cv. Robothave been established and propagated on medium containing yeastextract. These cultures consisted of unorganized callus tissuebearing embryogenic centres which increased in size during subculture,yielding new regenerated somatic embryos at the end of each20-d subculture. A development in the propagation of the embryogenic cultureswas the establishment of single embryo culture in hormone-freemedium where, in selected cases, the process of recurrent somaticembryogenesis (RSE) took place on the hypocotyl of explantedembryos. The process was independent of supporting callus tissueand occurred on simple defined medium. Single embryos underwenteither plantlet development or continued RSE on the hypocotyl.One third of the regenerated plantlets showed RSE after thetwo to three trifoliate leaf stage. In these cases shoot developmentstopped and only somatic embryo production took place. In vitrocloning of regenerated plantlets allowed us to reproduce eachparticular genotype before transplantation into soil. Lucerne (alfalfa), Medicago sativa L., somatic embryogenesis, single embryo culture     

Trade Winds Reduce Growth and Influence Gas Exchange Patterns in Papaya Seedlings

Four experiments were conducted to determine the effect of tradewinds in Guam, USA, on growth and gas exchange of three papaya(Carica papaya L.) cultivars. ‘Known You 1’, ‘Sunrise’,and ‘Tainung 2’ papaya seedlings at two differentstages of development were exposed to 0, 36 or 100% ambientwind. Wind exposure reduced stem height and leaf or stem dryweight in most cases, but had little influence on root growth.Net CO₂assimilation (A_CO2) at midday was lower for seedlingsexposed to wind than for those protected from wind. Dark respirationof exposed seedlings increased as much as 120% above that ofthe protected seedlings during the night. Wind exposure decreasedwhole plant evapotranspiration by up to 36% during the photoperiod,but increased evapotranspiration by 58–87% during thenocturnal period. Responses to wind exposure were similar amongcultivars, except that growth of ‘Tainung 2’ seedlingswas less affected by wind than that of the other cultivars.Seedlings that were exposed to the various wind treatments fromgermination onwards were less influenced by wind exposure thanwere seedlings that were grown in a protected nursery beforebeing exposed to the various wind treatments. These data indicatethat: (1) ambient trade winds in Guam are strong enough to decreasethe growth of papaya seedlings; (2) plant age influences theresponse; (3) stem and leaf growth are more influenced thanroot growth; and (4) decreasedA_CO2 and increased dark respirationmay be partly responsible for growth reduction. Copyright 2001Annals of Botany Company Carica papaya, gas exchange, wind     

A protocol for efficient transformation and regeneration of Carica papaya L.

Summary A reproducible and effective biolistic method for transforming papaya (Carica papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors in this protocol included: 1) spreading of young somatic embryo tissue that arose directly from excised immature zygotic embryos, followed by another spreading of the actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of kanamycin selection from all subsequent steps after kanamycin-resistant clusters were first isolated from induction media containing kanamycin; 3) transfer of embryos with finger-like extensions to maturation medium; and 4) transferring explants from germination to the root development medium only after the explants had elongating root initials, had at least two green true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 transgenic papaya lines expressing the nontranslatable coat protein gene of papaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. This also represents an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated this procedure in our laboratory by visiting researchers who did four independent projects to transform seven papaya cultivars with coat protein gene constructs of PRSV strains from four different countries. The method is described in detail and should be useful for the routine transformation and regeneration of papaya. Based in part on a presentation at the 1997 SIVB Congress on In Vitro Biology held in Washington, DC, June 14–18, 1997.     

In Vitro Embryo Culture and Induction of Multiple Shoots in Lychee (Litchi chinensis Sonn.)

Immature embryos of different sizes and ages from commercialvarieties of lychee (Litchi chinensis Sonn.) were cultured ina range of different media. Embryos as small as 3 mm could becultured using in vitro techniques and subsequently grown intoplants. MS solid medium with 2% sucrose supplemented with 150ml l^–1 coconut water was most effective in stimulatingthe germination of immature lychee embryos. Embryos of lycheewere treated to induce adventitious buds from embryonic shootsas a means of achieving multiplication. The different varietiesexhibited differences in response, with Bengal embryonic shootsproducing 15 adventitious buds after pretreatment with 100 mgl^–1 BAP for 3 h. Root formation was achieved in 65% ofadventitious shoots using MS medium supplemented with 0.5 mgl^–1 NAA and activated charcoal. These plants were successfullydeflasked and grown on in the glasshouse. This technique providesof means of producing some multiple shoots from lychee embryosand has value for multiplication in a breeding program wherea method of micropropagation is unavailable. Litchi chinensis Sonn., lychee, embryo culture, multiple shoots, in vitro     

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

Regulation of Soybean Embryogenesis by Abscisic Acid

Abscisic Acid (ABA) stimulates growth and protein accumulationin soybean (Glycine max. L. Merr.) embryos during the earlyphases of embryogenesis. Growth of mid-stage embryos is suppressedby ABA, but protein accumulation is not impaired. Metabolitedistribution studies indicate that ABA alters partitioning ofsucrose in older embryos such that protein accumulation is sustainedat the expense of lipid accumulation. The responses of in vitrocultured embryos to ABA is consistent with the normal patternof ABA accumulation and disappearance that occurs during embryogenesisin situ. A close correlation exists between ABA levels and embryogrowth rates in situ in three cultivars of soybeans. Dependingon the age or stage of the developing embryo, ABA either servesto promote or inhibit embryo growth. Key words: Embryogenesis, ABA, Seeds, Soybean.     

Adventitious Bud Formation from Mature Embryos of Picea chihuahuana Martinez, an Endangered Mexican Spruce Tree

Zygotic embryos of Picea chihuahuana Martínez were cultivatedin vitro to determine the time of organogenic competence andto maximize adventitious bud induction. The induction mediumconsisted of modified B5 substrate supplemented with N⁶-benzyladenine(with or without naphthalene acetic acid) or kinetin (with orwithout 2-4, dichlorophenoxyacetic acid) at different concentrationsand induction times. The minimum induction time required forbud formation was 14 d with kinetin and 17 d with N⁶-benzyladenine.After induction embryos were transferred to the proliferationmedium (modified B5 substrate with 50% of its components andwithout growth regulators) for 30 d. The subsequent buds weretransferred every 15 d to Schenk and Hildebrandt medium at halfits concentration without growth regulators. The most effectivetreatments were 3 and 5 mg l^-1kinetin or N⁶-benzyladenine whichproduced five to seven buds per embryo. The largest shoots weresubjected to rooting trials with pulses of different concentrationsof indole butyric acid resulting in only one bud developinga root. Histological analysis revealed clusters of three tofour cells that became more evident as induction time increased.Kinetin promoted the development of an organized structure priorto adventitious buds formation sooner than N⁶-benzyladenine.Copyright 2000 Annals of Botany Company Competence, plant tissue culture, micropropagation, Picea chihuahuana, endangered species, spruce     

Potential for introducing cold tolerance into papaya by transformation with C-repeat binding factor (CBF) genes

Papaya (Carica papaya L.) production is affected by low temperatures that occur periodically in the subtropics. The C-repeat binding factor (CBF) gene family is known to induce the cold acclimation pathway in Arabidopsis thaliana. Embryogenic papaya cultures were induced from hypocotyls of “Sunrise Solo” zygotic embryos on semisolid induction medium. The CBF 1/CBF 3 genes along with the neomycin phosphotransferase (NPT II) gene were placed under the control of the CaMV 35 S promoter and introduced into a binary vector pGA 643. Embryogenic cultures were transformed with Agrobacterium strain GV 3101 harboring pGA 643. After selection of transformed embryogenic cultures for resistance to 300 mg l⁻¹ kanamycin, somatic embryo development was initiated and transgenic plants were regenerated. The presence of the CBF transgenes in regenerated plants was confirmed by Southern blot hybridization. The papaya and the related cold-tolerant Vasconcella genomes were probed for the presence of cold inducible sequences using polymerase chain reaction (PCR). Possible cold inducible sequences were present in the Vasconcella genome but were absent in the Carica genome.     

Effects of Desiccation and Cryopreservation on the In Vitro Viability of Embryos of the Recalcitrant Seed Species Araucaria hunsteinii K. Schum

Embryos excised from fresh seed had a moisture content of about37% (fr. wt.), 7% above that of the whole seed: mainly as aresult of the dryness of the seed coat. When cultured for 14d at 26°C embryo growth ranged from unorganized to fullyorganized. Embryos which had been directly surface-sterilizedexhibited lower fully organized growth than aseptically excisedembryos. During desiccation of the whole seed at 15°C and 15% r.h.moisture loss from the embryo was slow until the embryo reacheda mean critical moisture content for fully organized growthin vitro of about 30%. After this point the rate of moistureloss increased, quickly diminishing the moisture content differentialbetween the embryo and the whole seed. No such shoulder in theembryo desiccation curve was observed when using a low viability(7%) seed batch. When dried below 30% moisture content, suchaseptically excised embryos increasingly exhibited unorganizedgrowth in vitro, with only root meristem survival (46%) at 13%moisture content. In contrast, a much higher level of root meristem survival (84%of control) was observed when aseptically excised embryos wererapidly desiccated to 13% moisture content, using a flow ofsterile air. However, the benefits of rapid drying were lostif the embryos were directly surface-sterilized prior to desiccation.Furthermore, 80% of aseptically excised embryos which survivedrapid desiccation to 20% moisture content exhibited root meristemsurvival following subsequent cryopreservation. These findingshave implications for the in vitro conservation of recalcitrantseed tissue. Key words: Araucaria hunsteinii K. Schum., recalcitrant seed embryo, desiccation, cryopreservation     

Effect of In Ovulo Period on the Differentiation and Regulation of Immature Embryos ofZamia Cultured In Vitro

Monnier, M. and Norstog, K. 1986. Effect of in ovulo periodon the differentiation and regulation of immature embryos ofZamia cultured in vitro—J. exp. Bot. 37: 1633–1642. Depending upon excision chronology, undifferentiated embryosof the cycad Zamia exhibited two types of development when culturedin vitro. (1) Early embryos, excised in July shortly after fertilization,showed considerable growth of suspensors but, at first, theembryos at the distal ends of the suspensors remained unchanged.As culture progressed, these embryos grew isodiametncally withoutorganogenesis. (2) Late embryos, excised in September, althoughalso undifferentiated at the time of explantation, directlyentered a period of organogenesis with rapid initiation of twocotyledons. These embryos, therefore, seemed to have receivedan inductive stimulus during their more prolonged stay in theovule which permitted subsequent differentiation. This organogeneticimpulse was also expressed even when the embryo was longitudinallybisected. Each half-embryo regenerated a complete embryo havingtwo cotyledons. In this instance, the independent developmentof each half-embryo was a phenomenon of regulation. However,when the longitudinal bisection was done on an old and well-differentiatedembryo, which already possessed two cotyledons, each half-embryocontinued to develop as if it remained attached to the missinghalf. Thus well-advanced embryos did not exhibit regulationto the same degree as do younger embryos and bisection of theembryo resulted in the formation of two half-embryos. Key words: Regulation, differentiation, embryo culture, Zamia     

Plant regeneration from Carica protoplasts

Summary Rapidly proliferating and highly regenerable suspension cultures of somatic embryos of Carica papaya x C. cauliflora were used for protoplast isolation. On average, protoplast yield was 1.5×10⁶/g fresh weight of somatic embryos. Protoplasts were first cultured in liquid KM8P-S medium for 2 weeks and then plated in the same medium solidified with 1% agarose. About 1.4% of the protoplasts developed directly into somatic embryos. Protoplast-derived somatic embryos proliferated rapidly through direct embryogenesis on modified MS medium supplemented with 1 mg/1 ABA, and developed into plantlets upon transfer to MS medium devoid of plant growth regulators. The plantlets were successfully transplanted to soil.Abbreviations MS Murashige and Skoog medium (1962) - KM8P Kao and Michayluk medium (1975) - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - CPW Frearson et al. medium (1973)     

Normalizing Development of Cultured Triticum aestivum L. Embryos: I. LOW OXYGEN TENSIONS AND EXOGENOUS ABA

Wheat (Triticum aestivum L.) embryos form in dynamically-regulatedovular environments. Our objectives were to improve developmentof cultured immature wheat embryos by simulating, in vitro,abscisic acid (ABA) levels and O₂ tensions as found in wheatovules during zygotic embryogenesis. We characterized from intactwheat kernels embryo respiration, embryo morphology and embryoand endosperm + ABA levels at 13, 19 and 25 d post-anthesis(DPA). Young (13 DPA) embryos were then excised and culturedin vitro, where they were exposed to 0·2 or 2·Ommol m^–3 ±ABA and 2.·1, 2·5 or 7·4mol m^–3 (6, 7 and 21%, respectively) gaseous O₂. At 6and 12 d in culture, + ABA levels, embryo respiration and embryomorphology were characterized by treatment. Thirteen-day-oldembryos from two different plant populations differed by 17-foldin initial ABA content. However, this difference did not affectprecocious germination in vitro, nor did it affect the amountof exogenous ABA required to reduce precocious germination by40%. In this respect, embryos from both populations were equallysensitive to exogenous ABA. Cavity sap O₂ levels (2·1to 2·5 mol m^–3) were much more effective in preventingprecocious germination of cultured embryos than were cavitysap levels of ABA (0·2 to 2·0 mmol m^–3).The combination of physiological levels of both ABA and O₂ largelynormalized DW accumulation and embryo morphology without alteringendogenous + ABA levels. Residual respiration of cultured embryoswas higher than that of embryos grown in situ, and was not influencedby the exogenous O₂ and ABA treatments Key words: Abscisic acid, embryo development, oxygen tensions, respiration, wheat     

Somatic Embryo Formation From Unemerged Inflorescences and Immature Embryos of a Graminaceous Crop Echinochloa

Formation of somatic embryos was dependent on concentrationof specific auxin and mineral nutrient formulation. On N₆ mediumwith low levels of 2,4-D somatic embryos were obtained fromunemerged inflorescences and immature embryos. Direct differentiationof somatic embryos, a rare feature of regeneration in graminaceousplants, was more apparent from immature embryos than from inflorescences.On the other hand, on MS medium with different levels of 2,4-Dcompact callus-like masses appeared which regenerated to formplantlets on auxin-free medium. At higher levels of 2,4-D andalso on N₆ medium compact tissues (morphogenic calli) appearedwhich were made up of thallus-like structures. Echinochloa, immature embryo, unemerged inflorescence, somatic embryo