Plant Cell, Tissue and Organ Culture 66: 121–131, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Somatic embryogenesis and long term maintenance of embryogenic linesfrom fox grapes

S. Y. Motoike, R. M. Skirvin∗, M. A. Norton & A. G. OtterbacherUniversity of Illinois, Department of Natural Resources and Environmental Sciences, 258 EDMML, 1201 W.Gregory Dr., Urbana, IL 61801 USA (∗requests for offprints: Fax: +1-217-333-477; E-mail: skirvin@uiuc.edu)

Received 21 July 2000; accepted in revised form 21 March 2001

Key words: charcoal, embryo conversion, embryo germination, embryo regeneration, Fredonia, grapes, Niagara,polyethylene glycol, Vitis×Labruscana L.H. Bailey

Abstract

In the present paper, a method for the induction and long-term maintenance embryogenic cultures for Vitis ×Labruscana ‘Niagara’ and ‘Fredonia’ is reported. Embryogenic cultures from these two cultivars were induced inan embryogenesis establishment medium (CIM) from ovaries obtained from flowers 10–14 days pre-anthesis. Theembryogenic lines obtained in this experiment have been stably maintained for more than 2 years, through repeatedsubcultures on a long-term maintenance medium (LTMM) without loss of embryogenic competence. Somaticembryo regeneration and maturation have been successfully achieved after 30 days of cultivating embryogeniccultures in an embryo regeneration medium (EDMM), supplemented with charcoal and polyethylene glycol. Thesomatic embryos were successfully germinated in two different media, ‘Fredonia’ germination medium (FGM) and‘Niagara’ germination medium (NGM), and converted into normal looking plants on a conversion medium (CM).

Introduction

Successful embryogenesis and subsequent mainten-ance and plant conversion in grapevines has beenrestricted to a few species including Vitis vinifera,V. longii and V. rupestris, or hybrids composed atleast for one of these species (reviewed by Grayand Meredith, 1992; Reisch and Pratt, 1996). Inthe last decade V. rotundifolia (Robacker, 1993), V.berlandieri×V. riparia (Mauro et al., 1995), V. latifo-lia (Salunkhe, 1999), and V. riparia (Xue et al., 1999)were added to the list, but not V.×labruscana L.H.Bailey. Nakano et al. (1997) attempted to make em-bryogenic cultures of V.×labruscana, but were com-pletely unsuccessful. Kikkert et al. (1997) reported0.1–3% embryogenesis in V.×labruscana cultivars,but made no mention of maintenance or conversion oftheir cultures to plants.

Many grape geneticists agree that most efficientway to genetically transform grapevines involves so-matic embryogenesis (Perl and Eshdat, 1998). Re-cently, many grapevines cells have been genetically

transformed and plants regenerated using somatic em-bryogenic cultures as the original target tissue; mostof them are, V. vinifera cultivars (Perl and Eshdat,1998). However, due to high genotype dependence,some grape species and cultivars remain recalcitrantto the process of embryogenesis and transformation.Vitis×labruscana cultivars have been among the mostrecalcitrant. This paper reports a method to induce andmaintain embryogenic cultures for two V.×labruscanacultivars (‘Fredonia’ and ‘Niagara’). We further re-port that these lines can be maintained for at least2 years, and that they could be readily converted toplants during that period.

Materials and methods

Culture initiation

To establish embryogenic cultures, flowers ofV.×Labruscana ‘Niagara’ and ‘Fredonia’ were col-lected 10–14 days before anthesis from the Univer-

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Figure 1. Explanted grape ovaries developing different types of callus after 50 days culture in CIM. (top) Embryogenic callus; (bottom)non-embryogenic callus.

sity of Illinois vineyards and stored in plastic bagsat 4◦C for approximately 72 h. Prior to dissection,flowers were disinfected with a 1.3% sodium hypo-clorite (NaOCl) solution containing 0.1% (v/v) Tween20 for 10 min and rinsed 4 times with sterile dis-tilled water (5 min/rinse). The ovary and its associatedtissues (receptacle + tissues attached to the ovary)were transferred to culture initiation medium (CIM).CIM was a modified version of Perl et al. (1995)embryogenesis reinitiation medium. It consisted ofNitsch and Nitsch (1969) salts supplemented with 30g l−1 sucrose, 100 mg l−1 myo-inositol, 0.8 g l−1

casein hydrolysate, 17 µM indole-3 acetyl-L-asparticacid (IASP, dissolved in dimethyl sulfoxide), 9 µM2,4-dichlorophenoxyacetic acid (2,4-D) and 1 µM 6-benzyladenine (BAP). The medium pH was adjustedto 5.6 prior to autoclaving and 2.5 g l−1 Phytagelwere added as the gelling agent. The medium wasautoclaved for 20 min at 121◦C. 2,4-D and BAP wereadded before autoclaving and IASP was added afterautoclaving. The medium was dispensed into 100×15-mm disposable Petri plates (30 ml per plate). Twenty-five detached ovaries and their receptacle tissues wereexplanted to each plate in a total of four replicated

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Figure 2. Increase of ‘Niagara’ and ‘Fredonia’ embryogenic culturediameter (mm) after 50 days of culture under different treatments,measured at the surface of the media where the cells had been incontact.

plates per cultivar. These plates were then incubatedin the dark at 21±1 ◦C until callus developed (ca. 50days). To minimize loss of explants due to contamina-tion, infected explants were eliminated from the Petridishes by cutting out the gel surrounding the explantsand lifting them from the plates as soon as they wereobserved.

Long term maintenance of embryogenic lines

To maintain long-term embryogenic lines, proem-bryonal masses obtained in CIM were cultivated ina long-term maintenance medium (LTMM). To de-velop LTMM, the effects of combinations of threegrowth regulators, IASP, 4 µM; 2,4-D, 2 µM; (1,2,3,-thiadiazol-5-yl)-N′-phenylurea (TDZ), 0.2 µM wasstudied in a randomized complete block experiment ina 2×2×2 factorial with four replications. All factorsother than growth regulators were as described forCIM. Each block included eight different treatments,

each in a different Petri dish. Five inocula consist-ing of proembryonic masses, approximately 2.0 mmin diameter, were plated in each Petri dish. After 50days of incubation under the environmental conditionsdescribed previously, the experiment was evaluated.The efficiency of each treatment was determined bymeasuring the diameter of the callus at the surfacewhere the cells had been in contact with medium. Themeans of each treatment were submitted to analysis ofvariance and tested by the t test (Steel et al., 1997).

Embryo development and maturation

The proembryonic masses proliferated in LTMM wereregenerated in an embryo development and matura-tion medium (EDMM). EDMM contained the sameminerals and organic nutrients as CIM but alteredgrowth regulators. EDMM included 17 µM IASP,10 µM β-naphthoxyacetic acid (NOA), 1 µM TDZ,and 1 µM abscisic acid (ABA). Also, three differentfactors in the medium were tested: 2.5 g l−1 activatedcharcoal (Darco S 51), 50 g l−1 polyethylene glycol(PEG, Amresco-OH), and 30 vs. 60 g l−1 sucrose. pHadjustments and Phytagel additions, as well as prepar-ation, autoclaving and dispensing were as describedpreviously. The plated proembryonic masses were in-cubated in a culture room maintained under 16-h days(cool white fluorescent light) between 20 and 22◦C.The photosynthetically active radiation (PAR) at thelevel of the medium surface was 131 µmol m−2 s−1.The experimental design was a randomized completeblock in a 2×2×2 factorial, with four replications.Each block contained eight different treatments andeach treatment was plotted in a separate Petri dish.Five randomly selected inocula consisting of proem-bryonic masses (ca. 2.0 mm in diameter) were platedin each Petri dish. After 30 days of incubation, thenumber of mature embryos in each callus was coun-ted. The means of each treatment were submitted toanalyses of variance and tested by the t-test (Steel etal., 1997).

Germination and conversion

The germination of mature somatic embryos was ac-complished in two different media, ‘Fredonia’ ger-mination medium (FGM) and ‘Niagara’ germinationmedium (NGM). FGM contained Nitsch and Nitsch(1969) salts, Staba (1969) vitamins, 30 g l−1 sucrose,100 mg l−1 myo-inositol, 0.4 µM BAP, 0.5 µM α-naphthalene-acetic acid (NAA), and 2.5 g l−1 Phyta-gel. pH was adjusted to 5.6 prior to autoclaving.

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Table 1. Summary of analysis of variance of the growth of ‘Niagara’ embryogenic culture after 50 daysof culture under different treatments, measured at the surface of the media where the cells had been incontact

Source df Mean square F Significance

TDZ (A) 1 59.1328 2860.8531 ∗∗IASP (B) 1 0.0153 0.7408 ns

2,4-D (C) 1 59.1328 2860.8531 ∗∗A×B 1 0.0153 0.7408 ns

A×C 1 59.1328 2860.8531 ∗∗A in C1 1 0.0000 0.0000 ns

A in C2 1 118.2656 5721.7063 ∗∗C in A1 1 118.2656 5721.7063 ∗∗C in A2 1 0.0000 0.0000 ns

B×C 1 0.0153 0.7408 ns

A×B×C 1 0.0153 0.7408 ns

Error 21 0.0207

∗∗Significant at p<0.01 by F -test; ns, non-significant.

Table 2. Summary of analysis of variance of the growth of ‘Fredonia’ embryogenic culture after 50 daysof culture under different treatments, measured at the surface of the media where the cells had been incontact

Source df Mean square F Significance

TDZ (A) 1 0.0703 0.4510 ns

IASP (B) 1 0.4753 3.0487 ns

2,4-D (C) 1 363.8253 2333.5937 ∗∗A×B 1 0.2628 1.6857 ns

A×C 1 0.0703 0.4510 ns

B×C 1 0.4753 3.0487 ns

A×B×C 1 0.2628 1.6857 ns

Error 21 0.1559

∗∗ Significant at p<0.01 by F -test; ns, non-significant.

NGM had the minerals and organic compounds as inFGM, but differed in growth regulators. NGM con-tained 4 µM IASP and no cytokinin. The germinationtests were performed in a series of 100×15-mm dis-posable Petri dishes in replicated experiments (fourreplications). Thirty completely formed embryos wereharvested from EDMM and transferred to each plate.The plates were incubated in the dark for 7 days andthen transferred to light in a culture room maintainedat the conditions described previously. After 14 daysunder light, germinated embryos were transferred toconversion medium (CM). The cm contained 75%C2D salts (Chee and Pool, 1983), Staba (1969) vit-amins, 22.5 g l−1 sucrose, 100 mg l−1 myo-inositol,1 µM NAA, and 8 g l−1 agar (Sigma–Aldrich). pH

was adjusted to 5.6 prior to autoclaving and the me-dium dispensed into baby food jars (ca 9.0×5.0 cm).The germinated plantlets were allowed to grow in thismedium for 30 days and then transferred to soil in agreenhouse.

Results and discussion

Embryogenic culture establishment

Embryogenic cultures were successfully induced fromovaries extracted 10–14 days prior to anthesis in CIM.The proembryonic mass-like structures (PEMs) wereobserved 50 days after ovary culture initiation, forboth cultivars, ‘Niagara’ and ‘Fredonia’, at rates of 25

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Figure 3. Niagara long-term embryogenic culture growing in LTMM supplemented with 2,4-D.

and 16%, respectively. PEMs were slow growing, fri-able, white to dark, with a nodular texture (Figure 1).Contamination was responsible for the loss of manyexplants (20 and 24% for ‘Niagara’ and ‘Fredonia’respectively).

The establishment and maintenance of embryo-genic cultures in Vitis sp. is highly genotype-dependent and thus far has been confined to a fewspecies and their hybrids (Gray and Meredith, 1992;Reisch and Pratt, 1996). In V.×Labruscana severalauxins, 2,4-D, 3,6-dichloro-o-anisic acid (Dicamba,DIC), NOA, 4-amino-3,5,6-trichloropicolinic acid(Picloram, PIC) and 2,4,5-trichloro-phenoxyaceticacid (2,4,5T), in combination with cytokinins, N-(2-chloro-4-pyridyl)-N ′-phenylurea (CPPU) and TDZwere unsuccessfully tested to induce embryogen-esis from leaf and flower tissues (Nakano et al.,1997). Kikert et al. (1997) reported a low fre-quency embryogenesis (0.1–3%) in anther cultures ofVitis×Labruscana, after nearly 1 year from the ini-tiation in various combinations of auxins (2,4-D andNOA) and cytokinin (BA and CPPU).

The higher frequency and precocity of embryo-genesis in CIM represent an improvement over thoseobtained by Kikkert et al. (1997) in V.×Labruscana.These results may be credited to the synergistic ef-fect of IASP and 2,4-D in inducing embryogenesis ingrapevines. The synergistic effect of these two auxinswas previously reported by Perl et al. (1995), who usedthe same combinations of growth regulators to inducerecallusing in mature somatic embryos of V. vinifera.

Long term maintenance embryogenic culture

Although CIM was efficiently used to induce, em-bryogenesis in V.×Labruscana, the medium was in-adequate to maintain long-term embryogenic cultures,which turned dark and eventually died. Preliminaryworks indicated that level of growth regulators couldbe too high, inhibiting the growth of embryogenic cul-tures. In addition, the use of TDZ instead of BA couldinduce higher rates of cell division stimulating growthas observed on the complex hybrid ‘Melody’ (data notshown). Thus, lower levels of auxins (IASP and 2,4-D)and together with TDZ were tested.

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Figure 4. Behavior of ‘Niagara’ embryogenic callus grown in LTMM supplemented with TDZ.

After a few days in various LTMM, ‘Fredonia’and ‘Niagara’ embryogenic cultures developed glob-ular embryogenic masses. An analysis of 50-day-oldcultures showed that 2,4-D alone was the most im-portant growth regulator for maintenance of long-termembryogenic cultures (Figure 2) in both ‘Niagara’ and‘Fredonia’. 2,4-D was essential for proliferation butalso prevented precocious regeneration and germina-tion that could lead to the loss of embryogenic com-petence (Figure 3). The interaction 2,4-D × TDZ wassignificantly negative for cultivar ‘Niagara’ (Table 1)when compared with treatments containing only 2,4-D

or 2,4-D + IASP (Figure 2). The embryogenic culturegrown in TDZ-supplemented media differentiated andlost embryogenic competence (Figure 4). Many abnor-mal embryos developed in this medium, which ger-minated precociously yielding aberrant plantlets thatdid not survive. In ‘Fredonia’ no significant effects ofTDZ or its interactions with other growth regulatorswere observed (Table 2). The effects of IASP or itsinteractions with other growth regulators were not sig-nificant for both cultivars (Tables 1 and 2); however,

treatments which included 2,4-D + IASP yielded largerembryogenic colonies in ‘Fredonia’ (Figure 2).

Transformation requires a tissue culture systemwith a large number of transformable cells, which willretain the ability for regeneration for a period of timesuitable to perform all necessary transformation andselection treatments (Birch, 1997). In addition, trans-formants are more efficiently selected from highlysynchronized cultures (Nakano et al., 1994; Perl etal., 1996) because the sensitivity of grape embryo-genic tissues to selection agents varies in differentstages of embryo development. Synchronized long-term embryogenic cultures were obtained in the treat-ments which included 2,4-D alone or 2,4-D + IASP(‘Niagara’ and ‘Fredonia’) and 2,4-D + TDZ or 2,4-D

+ TDZ + IASP (only ‘Fredonia’).The roles of growth regulators in somatic embryo-

genesis have previously been studied. In grapevine,auxins, especially 2,4-D, have been very effective forinducing somatic embryogenesis (Gray and Meredith,1992), but can inhibit subsequent embryo develop-ment (Komamine et al., 1992). They have been re-ported to arrest the development of embryos at the

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Figure 5. Somatic embryo regeneration from 2.0-mm diameter ‘Fredonia’ (Fr) and ‘Niagara’ (Ni) embryogenic callus after 30 days culture inEDMM supplemented with charcoal.

globular proembryonic stage, and induce indefiniteproliferation of embryonic cells (Litz and Gray, 1992).In this research all V.×labruscana embryogenic lineshave been maintained up to 2 years, under frequentsubculturings in LTMM supplemented with 2,4-D,with no loss of embryogenic competence.

Cytokinin may increase the growth rates of PEMs(Komamine et al., 1992) and stimulate embryo devel-opment (Litz and Gray, 1992). Therefore the negativeinteraction between 2,4-D and TDZ in ‘Niagara’ em-bryogenic cultures may be due to the unsuitable ratioof these growth regulators for this cultivar.

Embryo development and maturation

In the present experiment, several factors weretested to induce the development and maturation ofV.×Labruscana somatic embryos. The results showedthat activated charcoal had a significant effect on em-bryo development (Tables 3 and 4). Proembryonicmasses regenerated somatic embryos when transferredto medium containing charcoal (Figure 5).

Activated charcoal can attenuate the deleterious ef-fects of phenolic compounds secreted from the planttissue during in vitro culture (Skirvin, 1981; Zhu et al.,1997). Several proteins have already been identifiedin grapevine embryogenic cultures, which impair thedifferentiation and development of somatic embryos(Coutos-Threvenot et al., 1992; Maes et al., 1997).Some of the purified antagonist proteins can be usedto maintain PEMs for transformation (Perl and Esh-dat, 1998). However, the use of charcoal in long-termmaintenance cultures may remove ingredients fromthe culture medium that are essential for maintenanceof PEMs.

The effects of PEG or sucrose alone or combin-ations of both, were not significant in embryo de-velopment and maturation of cultivars ‘Niagara’ and‘Fredonia’. However, a significant interaction betweenPEG and charcoal was observed for both cultivars(Tables 3 and 4). This interaction increased the yieldof mature embryos in ‘Fredonia’ and ‘Niagara’, twoand three times, respectively, when compared withthe yields obtained in the treatment that included onlycharcoal (Figure 6). No significant effect of the in-

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Table 3. Summary of analysis of variance of the number of ‘Niagara’ mature embryos developed from2.0-mm diameter embryogenic callus, after 30 days culture in EDMM under different treatments

Source df MS F Significance

Charcoal (CH) 1 49.4685 49.4410 ∗∗Sucrose (S) 1 0.0140 0.0140 ns

PEG 1 6.4597 6.4561 ∗CH×S 1 0.0269 0.0269 ns

CH×PEG 1 6.6946 6.6909 ∗CH in PEG1 1 9.8834 9.8779 ∗∗CH in PEG2 1 46.2797 46.2540 ∗∗PEG in CH1 1 0.0010 0.0010 ns

PEG in CH2 1 13.1533 13.1460 ∗∗S×PEG 1 0.5943 0.5940 ns

CH×S×PEG 1 0.5258 0.5255 ns

Error 21 1.0006

∗Significant at p<0.05 by F-test; ∗∗significant at p<0.01 by F-test; ns, non-significant.

Figure 6. Numbers of ‘Niagara’ and ‘Fredonia’ mature embryosdeveloped from 2.0-mm embryogenic callus, after 30 days culturein EDMM under different treatments.

teraction Charcoal × Sucrose was observed in thisexperiment.

The water relation between the embryo and itsenvironment, in vitro or in vivo, plays an importantregulator role in embryo development and matura-tion (Misra et al., 1985). Altering sucrose levels inthe medium has been sufficient to enable maturation.However, osmotica such as PEG or a combination ofsucrose and PEG in the medium have been more ef-fective (Etienne et al., 1993). In this experiment, PEGwas more effective for V.×Labruscana embryogeniccultures than sucrose.

Germination and conversion

Germination of somatic embryos is characterized bycotyledon expansion and chlorophyll formation, fol-lowed by radicle and hypocotyl elongation (Merkleand Wiecko, 1990). The process of shoot meristemdevelopment and subsequent vegetative leaf initiationfrom the apical meristem is known as conversion(Nickle and Yeung, 1994). In the present study, so-matic embryos of ‘Fredonia’ obtained from EDMMgerminated well in FGM (93%) but only 18% con-verted into normal plants with roots, shoots andleaves. Fewer ‘Niagara’ somatic embryos germin-ated in NGM (56%) and converted (15%). Many ofthe ‘Niagara’ and ‘Fredonia’ somatic embryos hadroots and cotyledons but failed to convert and somewere albino. ‘Niagara’ and ‘Fredonia’ plantlets havebeen moved to a greenhouse where they appear to begrowing normally (Figure 7).

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Table 4. Summary of analysis of variance of the number of ‘Fredonia’ mature embryos developed from2.0-mm diameter embryogenic callus, after 30 days culture in EDMM under different treatments

Source df Mean square F Significance

Charcoal (CH) 1 418.0693 260.1787 ∗∗Sucrose (S) 1 0.0038 0.0024 ns

PEG 1 19.5933 12.1936 ∗∗CH×S 1 0.0038 0.0024 ns

CH×PEG 1 19.5933 12.1936 ∗∗CH in PEG1 1 128.3253 79.8612 ∗∗CH in PEG2 1 309.3373 192.5111 ∗∗PEG in CH1 1 0.0000 0.0000 ns

PGE in CH2 1 39.1865 24.3871 ∗∗S×PEG 1 0.5366 0.3339 ns

CH×S×PEG 1 0.5366 0.3339 ns

Error 21 1.6069

∗∗Significant at p<0.01 by F -test; ns, non-significant.

Figure 7. Plants from converted V.×labruscana embryos growing in a greenhouse.

Good quality embryo production is important forincreased rates of germination and conversion, whichare limiting steps for a practical use of somatic em-bryogenesis of grapevine (Gray and Meredith, 1992).

The failure of germination and conversion of grapev-ine somatic embryos is the lack of a functional apicalshoot in mature embryos. Faure et al. (1996) observedthat the shoot apex is present in all somatic embryos at

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torpedo stage; however, the apical shoot differentiatesand loses its meristematic characteristics during devel-opment. Precocious germination generally occurs incultured embryos when the maturation phase has beendisturbed.

Conclusions

This is the first report of successful maintenanceand recovery of plants from embryogenic cultures ofV.×Labruscana. Proembryonic lines obtained in thisresearch are highly synchronized and each major de-velopmental stage (i.e., proembryo, mature embryoand germination) can be controlled by varying me-dia constituents. This control should facilitate therecovery of transformants efficiently.

Acknowledgements

This report was supported in part by funds providedby the University of Illinois College of Agriculture,Consumer, and Environmental Sciences (ACES) of-fice of Academic Programs, funds from University ofIllinois Agricultural Experiment Station project num-ber 65-0323, and funds from Brazilian Federal Agencyfor Post-graduate Education (CAPES) project numberBEX1386/96-4. Any opinions, findings, conclusions,or recommendations expressed in this publication arethose of the author(s) and do not necessarily reflect theview of the US Department of Agriculture.

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