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Inoculation of somatic embryos of sweet potatowith an arbuscular mycorrhizal fungus improvesembryo survival and plantlet formation

CARVALHO,C.H.S.

2000

Wellington Bressan, Carlos Henrique Siqueira de Carvalho, and David M. Sylvia

Abstract: Responses of somatic embryos of sweet potato (lpomoea batata (L.) Poir., cv. White Star) at different devel-opmental stages to in vitro inoculation with Glomus etunicatum (Becker and Gerdemann) (isolate INVAM FL329) wereevaluated. Somatic embryos were grown in glass tubes containing sterilized vermiculite and sand. A layer of natrosolplus White's medium was used as a carrier for arbuscular mycorrhizal (AM) fungal spores. Survival of embryos inocu-lated with AM fungi was significantly (P < 0.05) greater than that of noninoculated embryos at the rooted-cotyledonary-torpedo and rooted-elongated-torpedo developmental stages. Mycorrhizae significantly (P < 0.05) in-creased plantlet formation only when inoculation occurred at the rooted-elongated-torpedo developmental stage. Thegrowth stage at which the embryos were inserted into the glass tubes exerted a significant influence upon plantlet for-mation, and plantlet formation was further enhanced by inoculation with G. etunicatum. Plantlet formation was greatestat the rooted-elongated-torpedo stage. These results demonstrate that inoculation of somatic embryos with AM fungiimproves embryo survival and plantlet formation, and could enhance use of somatic embryos as synthetic seeds.

Key words: mycorrhiza, Glomus etunicatum, somatic embryos.

Résumé : Nous avons étudié Ia réponse d'embryons somatiques de Ia pomme de terre sucrée (lpomoea batata (L.)Poir., cv. White Star) à différentes étapes du développement suite à l'inoculation in vitro avec du Glomus etunicatum(Becker et Gerdemann) (isolat INVAM FL329). Les embryons ont été cultivés dans des éprouvettes contenant du sableet de Ia vermiculite stériles. Un enrobage avec du milieu White + Natrosol a servi à l'inoculation de spores du charnpi-gnon des mycorhizes des arbuscules (AM). La survie des embryons inoculés avec le champignon AM a été significati-vement plus élevée (P < 0.05) que celIe d'embryons non inoculés aux stades de développement des cotylédons quis'enracinent ou de croissance des racines allongées en processus d'enracinement continuo Les mycorhizes ont significa-tivement favorisé Ia formation de plantules mais seulement lorsque l'inoculation avait lieu au stade de Ia formation desracines allongées qui s'enracinent. Lors de Ia mise en éprouvettes, il a été constaté que le stade de croissance avait uneinfluence significative sur Ia formation de plantules et que celle-ci était favorisée par l'inoculation avec le G. etunica-tum. Le stade de développement des racines alIongées était celui qui permettait Ia meilleure production de plantules.Les résultats obtenus confirmem que l'inoculation dernbryons somatiques avec des champignons AM augmente Iasurvie des embryons et Ia formation des plantules et quil faudrait favoriser l'utilisation des embryons somatiquescomme semences synthétiques.

[Traduit par Ia Rédaction]

Mots clés : mycorhizes, Glomus etunicatum, embryons somatiques.

"-- The use of somatic embryos as "synthetic seeds" has po-tential applications in providing large-scale planting of out-standing genotypes of self-incornpatible species that aredifficult to propagate vegetatively, and for propagation of

Received December 20, 1999. Revision received April 5,2000. Accepted April 6, 2000. Published on NRC ResearchPress web site July 17,2000.

W. Bressan! and C.H. Siqueira de Carvalho. EMBRAPA /Embrapa Milho e Sorgo, Departrnent of Soil Microbiology,P.O. Box 151,35701-970, Sete Lagoas, MG, Brazil.D.M. Sylvia. Soil and Water Science Department, Institute ofFood and Agricultural Sciences, University of Florida,Gainesville, FL 32611-0290, U.S.A.

IAuthor to whom all correspondence should be addressed(e-rnail: bressan@cnpms.embrapa.br).

Cano J. Microbiol. 46: 741-743 (2000)

nove I genotypes produced by genetic engineering that arenot meiotically stable, and for maintenance of parental in-bred lines (Gray et aI. 1987). Germination in the soil re-quires the somatic embryos to be self-sufficient for root andshoot formation. Taylor (1986) suggested the use of gel ma-terial with a supply of nutrients for improved gerrninationand early seedling growth of zygotic seeded species.Schultheis et aI. (1988) reported that macronutrients addedto the gel increased the percentage of embryos rooted, andthat macronutrients and micronutrients are needed for opti-mum plant production. Beyond this, beneficial microorgan-isms like arbuscular mycorrhizal (AM) fungi can be addedto the gel material. Mycorrhizal studies have been performedin vitro using axenic plantlets grown frorn seed, and fungigrown in pure culture (Molina 1981; Blal et ai. 1990).Strullu and Romand (1987) reported that plants raised fromcoated somatic embryos were able to form in vitro typical

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742 Cano J. Microbial. VaI. 46, 2000

Table 1. Survival of inoculated and noninoculated sweet potato somatic ernbryos after 30 days of growth in steril-ized vermiculite and sand + Natrosol (2% solution) and White's medium. Results are means of 30 replicates.

Embryo type Noninoculated Inoculated with Glomus etunicatum

Embryo survival (%)*

Cotyledonary torpedoRooted-cotyledonary-torpedoElongated torpedoRooted-elongated-torpedo

0.0 Da16.5 Bb2.0 Ca

20.8 Ab

0.0 Da22.4 Ba

2.8 Ca36.9 Aa

*Means within a column followed by the same capital letter and within a row followed by the same lower case letter do not differsignificantly at P < 0.05 by Duncari's multiple range test. Significance deterrnined after are sine transformation.

Table 2. Mean plantlet formation and plantlet root colonization of sweet potato somatic embryos inoculated or uninoculated withGlomus etunicatum (INVAM FL329).

Plantlet formation (%)*

Plantlet root colonization (%)lnoculated with Glomus etunicatumEmbryo stage Noninoculated

Cotyledonary torpedoRooted-cotyledonary torpedoElongated torpedoRooted-elongated torpedo

0.0 Da2.5 Ca4.2.Ba15.6 Aa

0.0 Ca2.0 Ba3.8 Ba8.2 Ab

0.0 O8.6 C

15.2 B28.8 A

*Means within a column followed by the sarne capital letter and within a row followed by the same lower case letter do not differ significantly atP < 0.05 by Duncans multiple range test. Significance deterrnined after are sine transformation.

vesicular-arbuscular mycorrhizas when inoculated with veg-etative mycelium of Glomus sp. regenerated from sterilizedroot segrnents. The objective of this study was to investigatethe effect of mycorrhizal symbiosis on the development ofsomatic embryos of sweet potato.

The experiment was conducted at the Soil and Water Sei-ence Department, University of Florida. Rooted and un-rooted sweet potato (lpomoea batata (L) Poir.) (cv. WhiteStar), somatic embryos of cotyledonary-torpedo and elon-gated-torpedo shapes, were inoculated with or without disin-fected germinated spores of Glomus etunicatum etunicatum(Becker and Gerdemann) (INVAM FL329) to determine ef-fects on embryo survival, plantlet production, and mycor-rhizal root colonization.

Friable, embryogenic calli from Murashige and Skoog(1962) medium containing 1.0 ~ of 6-benzylaminopurinewere removed from plates and collected in beakers. Cell ag-gregates were gently broken apart using a glass slide, thenseparated into 335 and 71O um size fractions using steelsieves, The largest fraction was plated for embryo produc-tion in petri dishes (100 x 15 mm) containing Murashigeand Skoog (1962) solid medium lacking growth regulators.Plates were incubated in the dark for one week, followed bya 10/14 hour lighrJdark cycle (252 ~01·m-2.Çl) at 26 ± 2°Cfor 20 days, until normal embryos were obtained. Abnormalembryos, those fused with others or with rnisshapen scutellartissues, were discarded. Embryos selected at cotyledonary,rooted-cotyledonary, elongated-torpedo, and rooted-elongated-torpedo stages were placed into a layer of 2% solution ofNatrosol (Hung et a!. 1991), amended with White's (1963)medium and inoculated with 50 disinfested AM fungusspores in glass tubes (150 x 25 mm) filled with sterilizedvermiculite and sand. Spores were disinfested with a 10%bleach solution with I drop of Tween 20 for 5 min, andwashed tive times with sterilized water, There were 30 repli-cates of each treatrnent arranged in a completely randomized

designo Embryo survival (%), plantlet formation, and mycor-rhizal root colonization (%) were evaluated 30 days after theembryos were placed in the glass tubes. The roots were sep-arated from the embryos, and plantlets were gently washedto remove vermiculite and sand particles, and then trans-ferred to tissue-holding capsules. The roots were stained ac-cording to Phillips and Hayman (1970) and mycorrhizal rootcolonization was determined using the grid-line-intersectmethod (Giovanetti and Mosse 1980). Data were analizedusing the general linear model (GLM) procedure of SAS(SAS Institute Inc. 1985). Significance of treatments wereevaluated by Duncan's multiple range test (P < 0.05). Per-centage embryo survival and root colonization data were aresine transforrned prior to analysis.

Typical AM symbiosis was found on inoculated plantletsderived from somatic embryos, and no contamination wasobserved among the inoculated embryos or plantlets. Thestage of development of the somatic embryos also affectedembryo survival and mycorrhizal syrnbiosis. Mycorrhizalfungus colonization significantly (P < 0.05) improvedembryo survival at the rooted-cotyledonary-torpedo androoted-elongated-torpedo developmental stages (Table I).These responses are likely due to greater availability of nu-trients to the somatic embryos due to uptake of nutrientsfrom the media through AM fungal hyphae. Significant differ-ences (P < O.OS} in embryo survival between inoculated andnoninoculated somatic embryos occurred when inoculationtook place at more advanced developmental stages. Rooted-elongated-torpedo embryos had the greatest survival, whichindicates that these embryos are better able to adapt to newenvironmental conditions. Inoculation with G. etunicatumsignificantly enhanced plantlet formation from somatic em-bryos at the rooted-elongated-torpedo stage (Table 2). Em-bryos transferred to the glass tubes at the most advancedstage of maturation had the greatest plantlet formation. Thepresence of roots in cotyledonary-torpedo embryos did not

© 2000 NRC Canada

Notes

enhance plantlet forrnation relative to that of the elongated-torpedo embryos, a more advanced stage of embryo matura-tion. This confinns that the stage of embryo maturation ismore important than the presence of raots to promoteplantlet forrnation, although the presence of roots could im-prove nutrient uptake fram the media and lead to greater em-bryo development and plantlet fonnation.

The percentage of plantlet roots colonized by mycorrhizalfungi ranged from 8.6% to 28.8% (Table 2). The highest col-onization was with rooted-elongated-torpedo embryos, anddecreased with less mature embryos. Somatic embryo re-sponse to mycorrhizal inoculation was dependent upondevelopmental stage, with embryos at later stages of matura-tion having highest survival, plantlet fonnation, and root col-onization of roots by AM fungi. Inoculation withmycorrhizal fungus was effective only at later embryonic de-velopmental stage, and only when roots were already pres-ent. We concluded that inoculation with AM fungi at the latestages of embryo development impraves survival andplantlet fonnation in vitro.

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