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Tissue Culture Response from SeedlingExplants of Commercial Barley CultivarsGrown in BulgariaNabil Abumhadi a , Kunka Kamenarova a , Georgi Dimov a , ElenaTodorovska a , Adelina Trifonova b , Kostadin Gecheff b & AtanasAtanassov aa AgroBioInstitute , 8, Dragan Tzankov Boulevard, 1164, Sofia,Bulgariab Plant Science Sweden AB , Herman-Ehles Vag 3–4, SE-26831, Svalov,SwedenPublished online: 23 Sep 2008.

To cite this article: Nabil Abumhadi , Kunka Kamenarova , Georgi Dimov , Elena Todorovska , AdelinaTrifonova , Kostadin Gecheff & Atanas Atanassov (2006) Tissue Culture Response from SeedlingExplants of Commercial Barley Cultivars Grown in Bulgaria, Journal of Crop Improvement, 15:1,51-65, DOI: 10.1300/J411v15n01_05

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Tissue Culture Responsefrom Seedling Explants

of Commercial Barley CultivarsGrown in Bulgaria

Nabil AbumhadiKunka Kamenarova

Georgi DimovElena TodorovskaAdelina TrifonovaKostadin GecheffAtanas Atanassov

ABSTRACT. Only a few studies have been conducted in which re-generability of barley has been examined. In the current study, 17 barleygenotypes (nine two-row malting type: Aster, Emon, Ruen, Jubiley,PV101, Korten, Krassi 2, Perun and Igri, and eight six-row feed types:Karnobat, Hemus, Jerun, Veslets, Aheloi 2, Diana, Panagon, and Izrgev)were evaluated for tissue-culture response from seedlings during athree-year period. Regenerable calli were obtained from all tested geno-types. Although there was much variation in regeneration capacityamong the cultivars, plants were obtained from all cultivars. The major-ity of green plants grown to maturity were fertile and normal in appear-

Nabil Abumhadi, Kunka Kamenarova, Georgi Dimov, Elena Todorovska, andAtanas Atanassov are affiliated with AgroBioInstitute, 8, Dragan Tzankov Boulevard,1164 Sofia, Bulgaria

Adelina Trifonova and Kostadin Gecheff are affiliated with Plant Science SwedenAB, Herman-Ehles Vag 3-4, SE-26831 Svalov, Sweden.

Address correspondence to: Nabil Abumhadi, AgroBioInstitute, 8, Dragan TzankovBoulevard, 1164 Sofia, Bulgaria (E-mail: nabumhadi@yahoo.com).

Journal of Crop Improvement, Vol. 15(1) (#29) 2005Available online at http://www.haworthpress.com/web/JCRIP

2005 by The Haworth Press, Inc. All rights reserved.doi:10.1300/J411v15n01_05 51

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ance. The frequency of embryogenic callus induction and regenerabilitywas influenced by genotype and growth conditions of the donor plants.Genotype was the most important determinant of the in vitro response.The best in vitro performance, based on ability to form regenerable calliand to regenerate plants was observed for Ruen, Aster, and Emon. [Ar-ticle copies available for a fee from The Haworth Document Delivery Service:1-800-HAWORTH. E-mail address: <docdelivery@haworthpress.com> Web-site: <http://www.HaworthPress.com> 2005 by The Haworth Press, Inc. Allrights reserved.]

KEYWORDS. Hordeum vulgare L., seedlings, somatic embryogen-esis, plant regeneration

ABBREVIATIONS. CPS–number of regenerants per callus; 2,4-D–2,4-Dichlorophenoxyacetic acid; 2iP–6-(gamma, gamma-Dimethylallyl-amino)purine; IAA–Indole-3-acetic acid; RPC–number of regenerantsper induced callus; RPS–number of regenerants per seedling; TNR–number of regenerants per plate; TNSC–traits-number of seedlings pro-duced calli

INTRODUCTION

Plant in vitro culture is an excellent system for fundamental studiesand applied research in plant breeding and genetic transformation. Planttissue culture can be maintained and under defined conditions, it is pos-sible to demonstrate that individual cells are totipotent and have the ca-pacity to regenerate new plants. Such applications of in vitro techniquesare now well established for many dicotyledonous species, but mono-cotyledonous species are more difficult to culture in vitro and the devel-opment of efficient regeneration systems has been slow (Jähne-Gärtnerand Lörz, 1996).

Reviews from the late 1970s noted that no technology essential for invitro genetics had been satisfactorily established for cereal species ingeneral and barley in particular (King et al., 1978). More recently, re-search in plant regeneration by somatic embryogenesis has been carriedout in barley with variable efficiency from a variety of explants: apicalmeristems, seedling mesocotyl, immature embryos, mature embryos,immature ovary tissue, immature inflorescences, anther and isolatedmicrospores. Dale and Deambrogio (1979) conducted a study on the ef-ficiency and reliability of a range of barley explants. They concluded

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that immature embryo explants were good for callus induction and plantregeneration. It must, however, be pointed out that immature explantsfor in vitro culture are generally available only in restricted periods orseasons of the year. Therefore, it might be prudent to consider maturetissues as a possible source of explants for better organization/planningof experiments. But mature tissues are considered to be more recalci-trant to tissue culturing. Developmental progression has been limited tocultures capable of somatic embryogenesis and plant regeneration di-rectly from the explant or via a callus phase using immature embryos(Lührs and Lörz, 1987; Dahleen and Bregitzer, 2002) or microspores(Jähne et al., 1991; Li and Devaux, 2001). Nevertheless, these explantsrequire continuous growth of donor plants under controlled environmen-tal conditions, which necessitates protocols that are labor-, time- andspace-consuming. Some studies that used the greenhouse did not reportthe months in which the plants were grown. Dahleen (1999) comparedthe in vitro response of two barley cultivars, ‘Golden Promise’ and‘Morex’, grown under greenhouse and growth chamber conditions, withplanting date as a variable factor. The author concluded that the genotype,growth environment, and planting date had significant effects on greenplant regeneration from barley immature embryos. In that paper, Dahleen(1999) demonstrated that planting date significantly affected regenera-tion response from greenhouse-grown donor plants. In addition, therewas a strong genotype dependency for tissue culture and stable transfor-mation of barley, as demonstrated by the fact that more than one-half ofall the investigations (studies) reporting on these processes in barley usedthe cultivar Golden Promise (Dahleen, 1999, Sharma et al., 2004).

Genotype, size and age of the explant, growth conditions of the donorplants, and media composition strongly influence plant regeneration.The influence of the genetic background of the cultivar used in inducingtissue culture appears to be a key factor for successfully regeneratingcultures (Lupotto, 1984). The objective of this study was to determinewhether this regeneration system could be beneficial for a wide range ofelite Bulgarian cultivars, and to identify potentially highly regenerablecultivars for future transformation work.

MATERIALS AND METHODS

Plant Materials

Sixteen commercial barley cultivars currently grown in Bulgaria andone Germany cultivar were used as experimental material. Ten of the

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cultivars used in this study have not been previously evaluated for plantregeneration. We report here on the ability of nine 2-row cultivars(Ruen, Jubiley 100, PV 101, Krassi 2, Korten, Aster, Perun, Emon, andIgri–as a standard) and eight 6-row cultivars (Veslets, Karnobat, Hemus,Aheloi 2, Panagon, Diana, Jerun, and Izgrev) to induce callus and re-generate plants from seedlings.

Plant materials were kindly supplied by Prof. Mersinkov–Agricul-tural Institute, Karnobat, Bulgaria, and Prof. Gorastev–AgriculturalUniversity, Plovdiv, Bulgaria. A total of 16 Bulgarian cultivars of win-ter type were used. These cultivars differ in genetic background (parent-age) and usage (Table 1).

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TABLE 1. Description of 16 Bulgarian barley cultivars (genotypes).

Genotype Type Researcher

Perun 2- row Malting D. Velcheva–Agricultural Institute, Karnobat,

Bulgaria

Jubiley 100 2- row Malting Ch. Gorstev–Agricultural University, Plovdiv,

BulgariaPV 101 2- row Malting

Krassi 2 2- row Malting

Ruen 2- row Malting N. Mersinkov–Agricultural Institute, Karnobat,

BulgariaKorten 2- row Malting

Aster 2- row Malting

Emon 2- row Malting

Panagon 6- row Feed

Veslets 6- row Feed St. Zaprianov–Agricultural Institute, Karnobat,

BulgariaKarnobat 6- row Feed

Hemus 6- row Feed

Aheloi 2 6- row Feed

Jerun 6- row Feed

Izgrev 6- row Feed

Diana 6- row Feed T. Stefanov–Agricultural Institute, Karnobat,

Bulgaria

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Media and in vitro Culture Procedure

Cultures were established and plants regenerated following pub-lished procedures for seedlings (Vitanova et al., 1995). Dry seeds weresterilized using 70% ethanol for 1 min. Seeds were treated for 30 minwith 5% Domestos® and rinsed three times in sterile water. The isolatedmature embryos were placed (scutellum down) for one-month pre-cultivation at 24°C in darkness on a solid medium containing B5 saltsand vitamins (Gamborg et al., 1968), supplemented with 30 g l�1 su-crose and 8 mg l�1 2,4-D and 8 g l�1 agar. Explants (1 cm in length), in-cluding part of the coleoptile and the bases of the first 3-4 leaves withthe complete apical meristems, were cut with a blade and placed for cal-lus induction on BMR medium (MS salts and vitamins; Murashige andSkoog, 1962), supplemented with 30 g l�1 sucrose, 3.3 mg l�1 2,4-D,0.3 mg l�1 2ip, and 1.8 mg�1 IAA (Figure 1). These explants were cul-tured at 25°C in dim (≈ 25 µmol m�2 s�1) light with daylength of 12 h.Four weeks after initiation, all embryogenic calli (Figure 2) were trans-ferred onto a hormone-free medium (MS salts and vitamins, supple-mented with 30 g l�1 sucrose) for plant development. Cultures weregrown at 24°C in bright light (≈ 125 µmol m�2s�1) with daylength of 16h for 30 days. Green plantlets (Figure 3) were transferred to rooting

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FIGURE 1. Seedling explants (1 cm in length) cultured on callus induction me-dium

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medium (hormone-free callus induction medium) in bright light (Figure4). The pH of the media was adjusted to 5.8, and then the medium wassolidified with 0.3% (w/v) Gelrite and autoclaved (121°C, 20 min). Atotal of 100 mature embryos of each genotype was cultured per experi-ment. The chemicals used in this study were provided by Duchefa(Haarlem, The Netherlands).

Statistical Analysis. The influences of genotype (cultivar), year (re-flecting the possible differences in the environmental and experimental

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FIGURE 2. Embryogenic calli after 4 weeks of culture on BMR medium

FIGURE 3. Regenerated green plantlets after one month of culturing on hor-mone-free medium

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conditions), barley type (2- and 6-row) were analyzed separately foreach of the five studied traits–number of seedlings that developed calli(TNSC) per plate, number of calli per seedling (CPS) where for alldishes 20 seedlings per plate were used, number of regenerants (TNR)per plate, number of regenerants per seedling (RPS), and number ofregenerants per callus (RPC). The following three-factor nested fixed-effects model with interaction was used:

Yijkl = Ii + Tj + Cjk + ITij + eijkl,

where Y is the number of observed characteristics for the number orpercentage per Petri plate, I–effect of the i-th year (i = 1, . . . , 3), T-effectof the j-th barley type, e.g., with 2- or 6-row (j = 1, 2), C-effect of thek-th genotype presented within the corresponding barley type and IT isthe interaction of the year with the barley type. The general influence ofeach effect was tested via the F-test (Table 2). The significance of thedifferences between least-squares (LS) means was assessed via thet-test.

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FIGURE 4. Healthy and green rooted barley plants

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RESULTS

Average Values for the Traits

For the 255 Petri dishes studied, the average values of the observedcalli (TNSC), expressed as the total number of calli that were producedfrom all 20 seedlings per plate, was 5.73 (Table 3). The correspondingvalue for the number of calli per seedling was 0.29. The regeneration ac-tivity (TNR) measured as the total number of regenerants per plate was60.4. Corresponding regeneration ability, i.e., the total number of re-

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TABLE 3. Mean values for the traits: number of seedlings produced calli(TNSC), number of regenerants per callus (CPS), number of regenerants perplate (TNR), number of regenerants per induced callus (RPC) and number ofregenerants per seedling (RPS).

Trait Mean S.D. CV

TNSC Callusgenesis per dish 5.73 3.39 59.2

CPS Calli per embryo 0.29 0.17 58.6

TNR Regeneration per dish 60.42 47.66 78.9

RPS Regeneration efficiency 3.02 2.38 78.8

RPC Regeneration ability 9.93 5.57 56.1

TABLE 2. Probability (P) for significant influence (F-test) of the studied factorsyear (Y), genotype (G) for each barley type (T), and year by type interaction(Y � T) on studied traits, with corresponding coefficient of determination, R2.

Traits d.f. TNSC CPS TNR RPS RPC

Effect F P F P F P F P F P

Year (Y) 2 21.90 *** 21.90 *** 1.70 ns 1.70 ns 31.68 ***

TYPE (T) 1 571.85 *** 571.85 *** 669.84 *** 669.84 *** 158.42 ***

G of 2 R 8 111.96 *** 111.96 *** 95.19 *** 95.19 *** 17.55 ***

G of 6 R 7 6.80 *** 6.80 *** 28.92 *** 28.92 *** 50.43 ***

Y � T 2 1.17 ns 1.17 ns .530 ns .530 ns 7.52 ***

Remainder 234 R2 .870 .870 .875 .875 .758

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generants per induced callus (RPC), was 9.9. The mean for the fifth trait-regeneration efficiency (RPS), measured as the number of regenerantsper seedling, was 3.0. For all observed characteristics, the variation(CV) was relatively high and was in the range of 56 to 79%.

Influence of the Various Factors

The genotype, year and barley type showed significant influences oneach of the studied traits–TNSC, CPS, TNR, RPS and RPC. All the fac-tors together accounted for about 87% of the variation. Somewhatsmaller influence was found for the regeneration ability-RPC, i.e., 76%(Table 3).

Callusgenesis–the TNSC and CPS–was influenced significantly (P <0.001) by genotype, barley type and year, but not by any interactions.The main influence seemed to be that of barley type, followed by the ge-notype. The influence of the year was also significant.

The TNR was highly affected by the genotype and barley type withinthe studied barley cultivars. The influence of the year and the interac-tions were not significant.

The fifth trait–RPC–was affected significantly by genotype, barleytype and year and by year-by-barley type interaction. The influence ofthe barley type was the largest, followed by cultivars in the 6-rowgroup. The smallest effect was for year and interaction. The relativelysmall effect of the cultivars from the 6-row type represents a tendencydifferent from one to be expected on the basis of the first two traits.

Estimates of the Effects of the Factors

The Year Effect

The TNSC was the same for the first and third year of the study, about6.0 calli per plate, whereas for 1999 the average number was muchlower, 4.87 (Table 4). For TNR, however, the effect of the first twoyears, 1998 and 1999, was very similar, whereas lower number, almost56, was noted for the last year. Being non-significant, the year influencehere was of relatively low importance. The relation between TNSC andTNR showed that they were affected specifically by environmentalfactors.

In spite of lower number of calli in 1999, the number of RPC (regen-eration ability) was larger that those for the other two years. This differ-

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TABLE 4. Mean values (LS-mean ± SE) for influence of the studied factors ontraits, TNSC, CPS, TNR, RPS, and RPC.

Factors/Traits

N TNSC CPS TNR RPS RPC

Mean 255 5.62 0.08 0.28 0.004 58.74 1.10 2.94 0.06 9.80 0.18

Year

1998 85 6.00 0.14 0.30 0.007 60.73 1.91 3.04 0.10 9.22 0.31

1999 85 4.87 0.14 0.24 0.007 59.54 1.91 2.98 0.10 11.76 0.31

2001 85 5.99 0.14 0.30 0.007 55.95 1.91 2.80 0.10 8.41 0.31

Type

2 rows 135 7.53 0.11 0.38 0.005 87.24 1.51 4.36 0.08 12.05 0.25

4 rows 120 3.71 0.12 0.18 0.006 30.24 1.60 1.51 0.08 7.55 0.26

Genotypesof 2- Row

Geno 1 10Ruen

15 10.53 0.33 0.53 0.016 148.40 4.53 7.42 0.23 14.28 0.74

Geno 1 11Jubiley

15 7.33 0.33 0.37 0.016 102.60 4.53 5.13 0.23 14.80 0.74

Geno 1 12PV 101

15 8.67 0.33 0.43 0.016 66.27 4.53 3.31 0.23 7.63 0.74

Geno 1 13Krassi 2

15 3.40 0.33 0.17 0.016 51.80 4.53 2.59 0.23 17.18 0.74

Geno 1 14Korten

15 2.53 0.33 0.13 0.016 23.93 4.53 1.20 ca 9.86 0.74

Geno 1 15Aster

15 11.80 0.33 0.59 0.016 131.93 4.53 6.60 0.23 11.04 0.74

Geno 1 16Perun

15 3.93 0.33 0.20 0.016 49.40 4.53 2.47 0.23 12.72 0.74

Geno 1 17Emon

15 11.07 0.33 0.55 0.016 136.00 4.53 6.80 0.23 12.29 0.74

Geno 1 18Igri

15 8.53 0.33 0.42 0.016 28.07 4.53 3.74 0.23 8.67 0.74

Genotypeswith 6 -Row

Geno 2 20Veslets

15 3.47 0.33 0.17 0.016 34.07 4.53 1.70 0.23 9.39 0.74

Geno 2 21Karnobat

15 4.47 0.33 0.22 0.016 81.33 4.53 4.07 0.23 18.27 0.74

Geno 2 22Hemus

15 4.87 0.33 0.24 0.016 21.67 4.53 1.08 0.23 4.51 0.74

Geno 2 23Aheloi 2

15 3.67 0.33 0.18 0.016 9.80 4.53 0.49 0.23 2.61 0.74

Geno 2 24Panagon

15 2.87 0.33 0.14 0.016 14.67 4.53 0.73 0.23 4.96 0.74

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ential response probably resulted from the quality of the calli produced(friability and embryogenic characters–data not shown).

Barley Type Effect

Two-row type cultivars (malting) developed almost twice the num-ber of calli and regenerants (Table 4)–7.5 and 87.2 compared with the6-row type (feed type). For all traits, this tendency was quite apparent,well expressed, significant and considerable. Differences between thetwo types showed that the malting type was more amenable to in vitroculture than the feed type.

In vitro Response of the Malting Type Cultivars

Considerable variation was found for both numbers of calli andregenerants among the eight cultivars studied (Table 4). Three cultivarsthat had more than 10 calli were Aster (11.8), Emon (11.1), and Ruen(10.5). Medium callusgenesis group with about 7-8 calli per plate con-sisted of the cultivars Jubiley (7.3) and PV101 (8.7). The cultivars withthe lowest callusgenesis were Korten (2.5), Krassi 2 (3.4), and Perun(3.9). The standard cultivar Igri belonged to the last group.

The same grouping of the cultivars was found for TNR. For thecultivars Aster, Emon, and Ruen, the number of regenerants per platevaried from 131.9 to 148.4; for the second group, i.e., Jubiley and

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Factors/Traits

N TNSC CPS TNR RPS RPC

Geno 2 26Diana

15 3.73 0.33 0.19 0.016 28.07 4.53 1.40 0.23 7.36 0.74

Geno 2 27Jerun

15 4.33 0.33 0.22 0.016 45.67 4.53 2.28 0.23 10.60 0.74

Geno 2 28Izgrev

15 2.27 0.33 0.11 0.016 6.67 4.53 0.33 0.23 2.68 0.74

Year �Type

1998�2 45 8.07 0.19 0.40 0.010 90.11 2.62 4.51 0.13 10.90 0.43

1998�4 40 3.93 0.20 0.20 0.010 31.35 2.78 1.57 0.14 7.55 0.45

1999�2 45 6.64 0.19 0.33 0.010 88.76 2.62 4.44 0.13 14.99 0.43

1999�4 40 3.10 0.20 0.16 0.010 30.33 2.78 1.52 0.14 8.53 0.45

2001�2 45 7.89 0.19 0.39 0.010 82.84 2.62 4.14 0.13 10.27 0.43

2001�4 40 4.10 0.20 0.20 0.010 29.05 2.78 1.45 0.14 6.56 0.45

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PV101, it varied from 66.3 to 102.6; and for the group with the lowestcallusgenesis, i.e., Korten, Krassi 2 and Perun, TNR varied from 23.9 to51.8 regenerants per plate.

The grouping described above was somewhat changed in terms of re-generation ability (RPC). Some of the cultivars from high level ofcallusgenesis, Ruen and Emon, had relatively high regeneration ability.This high RPC group, however, contained cultivars with lower level ofcallusgenesis (Krassi 2 and Perun) as well as some from the mediumTNSC group, e.g., Jubiley.

In vitro Response of the Four-Row Forage Type Cultivars

Among the examined eight cultivars of the 6-row class, the variationin callusgenesis was between 2.3 to 4.9 (Table 4). The callusgenesis re-sponse was much lower compared with the 2-row class. The group witha relatively higher level of callusgenesis contained the cultivars Karnobat,Hemus and Jerun–above 4 calli per plate, followed by Veslets, Aheloiand Diana, with a range of 3.5 to 3.7 calli. Those with the lowest numberof calli were Panagon and Izrgev.

Some of cultivars with a high level of callusgenesis also had highlevel of regeneration potential (TNR); for example, Karnobat and Jerun.A similar result was found for the relatively low level callusgenesiscultivars, e.g., Izgrev.

The number of RPC varied relatively widely. It was high for cultivarswith a high level of callusgenesis, e.g., Karnobat, Jerun. A relativelylow level of RPC was found among all TNSC groups. Results suggestedthat within this group, cultivars could be selected for their regenerationability for both absolute and relative number of regenerants.

Year by Class Interaction (Year Type)

For all traits, the interaction of year and class type was not significant.It meant that the normal year fluctuations did not influence considerablythe regeneration ability and efficiency. Within each year, the 2-rowcultivars had in vitro response much better than that of the 6-row ones.

DISCUSSION

Coefficient of determination provided the evidence that the factorsbarley type, cultivar and year, but not interactions, accounted for the

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majority of the variation. Division of total number of calli and regen-erants by the number of embryos/seedlings per plate did not contributeto more accurate estimation of the influence of these factors because ofthe scaling effect. The identical figures for the variation of the first twotraits (TNSC and TNR) and the second two traits (CPS and RPS)showed that for their accurate characterization, only one of the twotraits is sufficient. Nevertheless, both sets of traits were used–the num-ber per Petri dish (first two traits) expressed the experimental conditionsand the second–per embryo allowed comparisons with other similarstudies, e.g., Castillo et al. (1998).

The most important factor that influenced the callus and regenerationability in the study was the type of cultivar. Two-row types were almosttwice more regenerable, as assessed by TNSC and TNR, than the 6-rowtypes. Similar tendency for a higher regeneration ability of two-rowbarley cultivars was found in other studies (Ohkoshi et al., 1991;Vitanova et al., 1995; Castillo et al., 1998). Consequently, in similarstudies twice more seedlings should be used for the higher row-typecultivars.

Variation among cultivars was significant for all traits. It was muchmore obvious for the 2-row type. For their regeneration ability, threesubgroups of cultivars were recognized and the lowest subgroup wassimilar to that of the six-row cultivars. It corresponded closely with pre-vious results from some of these cultivars (Vitanova et al., 1995). Theresults from the Bulgarian cultivars were similar to these from the litera-ture where marked between-cultivars variation was reported in embryo-genic callus induction and plant regeneration for barley cultivars adaptedto Central Europe, North America, Japan, and Spain (Hanzal et al., 1985;Lührs and Lörz, 1987; Bregitzer, 1992; Baillie et al., 1993; Ohkoshi etal., 1991; Castillo et al., 1998). The rate of callusgenesis and the regen-eration ability from barley seedlings were two independent processesthat were controlled by independent genetic systems (Komatsuda et al.,1989; Mano et al., 1996; Dahleen, 1999; Rikiiski and Yasuda, 1994;Abumhadi et al., 2005).

The year effect reflected both the experimental conditions and the en-vironment for development of the donor plants. For all traits related toregeneration ability, this effect was significant but not very strong.Other studies, on factors affecting regeneration from callus cultures de-rived from embryos, rarely mentioned donor plant growth environment(Dunwell, 1986; Mohan Jain et al., 1988; Jähne-Gärtner and Lörz, 1996).In general, the results of this study corresponded with others using a va-riety of cultivars, e.g., the study of the haploid production from anthers

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and microspores (Pickering and Devaux, 1992), with most emphasis ontemperature. Only a few studies described donor plant growth condi-tions (Baillie et al., 1993; Bregitzer, 1992; Dahleen, 1995; Goldsteinand Kronstad, 1986). Interaction effects of year and cultivar type werenot significant, which was an indication that donor plant environmentinfluenced the regeneration ability of the diverse barley types in a simi-lar way.

CONCLUSIONS

Regeneration ability of barley was affected most heavily by thecultivar type and by the cultivars within 2-row type. Ruen, Emon, andAster were the most appropriate cultivars for in vitro regeneration. Do-nor plant conditions influenced slightly the development of seedlings.No specific environmental effect was found for 2- and 6-row cultivars.

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