Plant Cell Reports (1984) 3: 125-129

Plant Cell Reports © Springer-Verlag 1984

Plant regeneration from mesocotyl callus of Hordeum vulgare L. Sibila Jelaska, Zdenko Rengel, and Vera Cesar Department of Botany, Faculty of Science, University of Zagreb, P.O. Box 933, 41001 Zagreb, Yugoslavia Received October 7, 1983 / Revised version received July 7, 1984 - Communicated by I. Potrykus

ABSTRACT

M A T E R I A L AND METHODS

Callus tissue was induced on barley mesocotyl explants of g e r m i n a t e d seven-day-old seedlings on MS m e d i u m supplemented with 2,4-D or 2,4,5-T in high concentrations. Two morphologically d i f f e r e n t tissue cultures were maintained in vitro for a long time: a callus tissue without o r g a n o g e n e s i s and a culture with high rhizogenic capacity. Shoots and plantlets were g e n e r a t e d when the a u x i n - m e d i a induced callus was transferred to m e d i u m supp l e m e n t e d with 3 ~M TIBA. In 62% of cultures, during the first five subcultures, four to t w e n t y e i g h t plants per single m e s o c o t y l were obtained. Some cultures produced shoots even in the 9th subculture, being in culture for nearly 14 months. The largest number of plants obtained per one m e s o c o t y l was forty. Plantlets rooted well on MS with 5.7 DM I ~ and survived t r a n s p l a n t a t i o n to soil in high percentages.

Seeds of the fourth g e n e r a t i o n of hybrids 4.36/3, 4.56/1, and 4.8/3 of two-rowed barley were used in these studies. Plant m a t e r i a l was s u r f a c e - s t e r i l i z e d as follows: after being scrubbed under running water with abrasive cloth (10 min), seeds were wetted with 95% ethanol (4 min), soaked in 3% (w/v) HalamJd a commercial product of N a - p - t o u l e n e s u l f o n chloramine, with a drop of d e t e r g e n t and continuous shaking (30 min) t and then rinsed in sterile d i s t i l l e d water (5 x 5 min). The sterilized seeds were g e r m i n a t e d in tubes on agar solidified 1/2 x Knop m e d i u m with 2% sucrose: 5 days at 4°C, and at room temperature (2024°C) during the sixth day. A second sterilization of the germinated seedlings was performed at the 7th day in the same way (with mechanical and ethanol treatment omitted) as with seeds, just before explants were taken. Explants were cultured on 15 ml agar m e d i u m in 30 x 160 mm tubes. D u r a t i o n of subcultures was for 4-6 weeks. A basal m e d i u m with macroand m i c r o e l e m e n t s according to M u r a s h i g e and Skoog (1962) supplemented with (mg.l-l) : 500 myo-inositol, 5 thiamine-HCl, 5 nicotinic acid, 5 pyridoxine-HCl, and 2-4.5% sucrose and 0.81% Difco Bacto-agar was used. For callus induction the growth regulators 2,4-D or 2,4,5-T were added to the media in various concentrations, and IAA, BA and TIBA were used according to the experimental objectives. M e d i a were sterilized by a u t o c l a v i n g at 121°C for 15 min. The cultures were incubated in a growth chamber at 26-27°C, exposed to an artificial light (day-light fluorescent tubes TE~-Zgb, 40W, 220V, 6.500K) of 500 ix (callus induction) or 1500-2000 ix (shoot regeneration) with lightdark cycles of 16/8 hours.

Abbreviations: IAA, i n d o l e - 3 y l - a c e t i c acid; BA, 6-benzylaminopurine; 2,4-D, 2 , 4 - d i c h l o r o p h e n o x y a c e t l c acid; 2,4,5-T, 2 , 4 , 5 - t r i c h l o r o p h e n o x y a c e t i c acid; TIBA, 2 , 3 , 5 - t r i i o d o b e n z o ic acid; MS, M u r a s h i g e and Skoog (1962) medium. INTRODUCTION C r e a t i o n and isolation of new genotypes by plant and tissue culture is possible, although their a p p l i c a t i o n to any breeding programs will depend on r e g e n e r a t i v e capacity and clonal f e a s i b i l i t y of certain economic species. Till now, barley (Hordeum vulgare L.), like other cereal crops, has not been an easy species for in vitro m a n i p u l a t i o n (Rice et al. 1978). This is caused by r e s t r i c t e d fund of cereal genotypes and routine approaches in the a p p l i c a t i o n of a standardized culture treatment. Recently, the m e t h o d using immature embryos has improved the tissue culture of cereals, and was used for tissue culture of barley, as well (Dale and D e a m b r o g i o 1979, 1980; Orton 1979). In this paper we report on e x p e r i m e n t s e s t a b l i s h i n g culture conditions for the induction and growth of callus as well as plant r e g e n e r a t i o n from tissue of m a t u r e embryos and seedlings of barley (Hordeum vulgare L.).

RESULTS Callus induction and growth For callus induction, d i f f e r e n t exp!ants (basal part of the first leaf, 1-2 mm mesocotyl with scutellum explant, and root w i t h tip meristem) were used. Tab. 1 shows frequency of callus i n d u c t i o n in r e l a t i o n to the type of explant and g r o w t h - r e g u l a t o r treatment. We used the two specific hormone c o n c e n t r a t i ons which had been successful a c c o r d i n g to

126 Cheng and Smith (1975) in barley tissue culture. The highest percentage of callus induction and the best growth c c u r r ~ on the mesocotyl explant. Induced ca]li ~ .... nodular, sometimes m o d e r a t e l y watery and pale yellow, with growth capacity after subculturing. Callus induction was compared using m e s o c o t y l explants placed on agar m e d i u m with 2,4-D + IAA scutellum side up and scutellum side down. There was a d i f f e r e n c e in the frequency of the explants producing callus, 70% and 100% respectively. Since the quality and v i a b i l i t y of induced callus tissue was better in the case of scutellum side down we used m e s o c o t y l explants placed on m e d i u m with the scutellum side down in all the following experiments. Table i. Callus induction on d i f f e r e n t explants of 7-day-old barley seedlings. Basal medium: m o d i f i e d MS + 3% sucrose + 0.8% agar. 24 explants were used per treatment.

Trea~_nt

Auxin }~M 2,4-D 45.2

2,4-D 15.8 + + IAA 11.4

Callus induction (% of cultured explants) Basal Mesocotyl ROot

Genotype

part

with

of the f~st leaf

scutell~

4.36/3 4.56/1 4.8/3

0 18 61

86 76 64

40 7 0

4.36/3 4.56/1

i0 0

85 73

ii 0

Looking for the optimal callus induction medium, we tested several c o n c e n t r a t i o n s of 2,4-D (13.5~ 22.6} 45.2 ~M), 2t4e5-T (11.7} 19.5} 31.3~ 39.1 ~M) and 2,4-D (15.8 ~M) in combination with IAA (11.4 ~M). Callus was induced in high percentages (75-100) of explants on all the tested c o n c e n t r a t i o n s of growth regulators, a l t h o u g h the increase of fresh weight of newly formed tissue was higher on lower auxin c o n c e n t r a t i o n s (Tab. i and 2). High c o n c e n t r a t i o n s of 2,4,D (36.1 and 45.2 DM) strongly inhibited rhizogenesis, which was not the case with 2,4,5-T. In Tab. 2 one can see that rhizogenesis occured even on the highest c o n c e n t r a t i o n of 2,4,5-T. The emergence of rootlets and calli c o m p r i s i n g green spots in primary cultures could be correlated w i t h the o c c u r r e n c e of shoot regeneration after t r a n s f e r r i n g tissue on fresh medium. The highest frequency of root and shoot forming cultures was o b t a i n e d on the m e d i u m with the addition of 19.5 ~M 2,4,5-T and 500 mg.l -I m y o - i n o s i t o l (Tab.2). The growth and o r g a n o g e n e s i s data o b t a i n e d in the p r e s e n c e and absence of m y o - i n o s i t o l to d i f f e r e n t concentrations of auxins are d i f f i c u l t to explain. We selected two kinds of tissue cultures capable of sustaining p r o l o n g e d growth. The first was a callus tissue w i t h o u t organogenic capacity and with good growth through 32 m o n t h s , o n the basal MS m e d i u m supplemented with i0 ~ M 2,4-D and 10 ~ 4 IAA (Fig.l). The

second, which had high rhizogenic capacity during many subcultures, grew on the basal MS m e d i u m with 3 ~M TIBA (Fig. 2). This line had no capacity of shoot regeneration. Pl__ant re~eneratio__~n In this investigation, however, p a r t i c u lar attention was paid to shoot and p l a n t l e t regeneration. For this purpose, calli induced in primary cultures were transferred onto several d i f f e r e n t m e d i a . T i s s u e s transferred to an auxin-free m e d i u m or a m e d i u m with 1 ~ M IAA and 1 ~M BA d e v e l o p e d lush roots in a high p e r c e n t a g e of cultures. Only one culture with r e g e n e r a t e d shoots was o b t a i n e d by these treatments. However, w h e n calli were transferred onto a m e d i u m supplemented with 3 D M TIBA, with a reduced c o n c e n t r a t i o n of sucrose (2%, w/v), cultures r e g e n e r a t e d shoots in 62.5%. A c c o r d i n g to Pental and Gunckel (1977) suggestion we counted only the cultures in which more than one plantlet occured. (Tab.3). During the first five subcultures 4-28 plants per single explant of m e s o c o t y l were o b t a i n e d (Tab. 3). Calli of the two mesocotyls continued to generate shoots (Figs. 3,4) to the 9th subculture (Tab. 3), being in culture for about 14 months. They gave a total of 23 and 40 plants. All shoots were green and we did not observe albinos. Shoots generated on m e d i u m with TIBA g e n e r a l l y did not root on this medium, a l t h o u g h the callus p r o d u c e d autonomous rootlets in the same cultures. C o m p l e t e plants with good root development were achieved by t r a n s f e r r i n g single shoots onto a m e d i u m supplemented w i t h 5.7 ~ M IAA (Fig. 5). Rooted plants (140 total) surv i v e d t r a n s p l a n t a t i o n to soil in very high percentage, being p r o t e c t e d from drying up during the first i0 days. They grew well and m o s t l y formed fertile heads. In these experiments we observed a decrease of shoot induction potential during the subculturing (Tab. 4). The same table shows that the m e d i u m supplemented with TIBA allowed d e v e l o p m e n t of n o t i c e a b l y more shoots than a m e d i u m with low c o n c e n t r a t i o n of 2,4,5-T. The parts of the same cultures were transferred on both media parallel. DISCUSSION Callus tissue was induced in m e s o c o t y l explants in high percentage with high concentrations of 2,4-D or 2,4,5-T (in range Ii.7-45.2 ALM) as well as on m e d i u m supplem e n t e d w i t h 2,4-D (15.8 ~M) and IAA (11.4 ~M). T h o u g h we did not go into histological inves t i g a t i o n s , it seems that callus was induced p r i m a r i l y on m e s o c o t y l tissue and very little, if at all, on scutellum. Saalbach and Koblitz (1978) induced callus on m e s o c o t y l explants with high c o n c e n t r a t i o n of 2,4-D (45.2 itM) too, and Dale and D e a m b r o g i o (1979) o b t a i n e d weak callus formation on m e s o c o t y l explants using a 10 times lower c o n c e n t r a t i o n . It may be that high c o n c e n t r a t i o n s of auxins are necessary for callus induction on barley mesocotyl. In order to achieve plant regeneration, barley calli had to be subcultured onto a reg e n e r a t i o n medium. The r e g e n e r a t i o n media were d i f f e r e n t both between themselves as well as from induction m e d i a (Cheng and Smith,

127 T a b l e 2. E f f e c t of a u x i n s (2,4-D and 2,4,5-T) on c a l l u s i n d u c t i o n , f r e s h w e i g h t and r h i z o g e n e s i s in p r i m a r y c u l t u r e of m e s o c o t y l e x p l a n t s and p e r c e n t a g e of c u l t u r e s w i t h shoot r e g e n e r a tion d u r i n g s u b c u l t u r i n g . B a s a l m e d i u m : MS + 4.5% s u c r o s e + 1% agar (genotype 4.36/3).

T R E A T M E N Ta Auxin myo-inositol DM m g . l -I

Cultures with callus induction %

Fresh weight b ~ ± SE

Primary cultures with rhizogenesis %

Cultures with shoot regeneration during 5 subcultures %

2,4-D 13.5 22.6 22.6 36.1 45.2 45.2

0 0 500 0 0 500

i00 100 100 100 100 75

69.3 144.8 94.7 50.3 59.7 53.3

10.3 32.7 14.9 4.2 7.3 4.3

0 25.0 12.5 0 0 0

12.5 25.0 12.5 0 12.5 0

0 0 500 0 0 500

i00 87.5 i00 i00 i00 i00

198.3 81.6 292.6 95.3 85 117.6

58 14 39 24 29 48

50.0 12.5 87.5 12.5 12.5 37.5

50.0 12.5 50.0 12.5 12.5 37.5

2,4,5-T 11.7 19.5 19.5 31.3 39.1 39.1

a8 e x p l a n t s bweighed

Table

after

treatment 33 d a y s

3. H i s t o r y 4.36/3/

C o d e of mesocotyl explant 1 2 3 4 5 6 7 8 9 i0 ii 12 13 14 15 16

per

in c u l t u r e

of shoot regeneration of barley.

Primary

culture a

Number

presented

for e a c h

of

16 m e s o c o t y l

explants

/ genotype

Auxin ~M

I.

of shoots r e g e n e r a t e d per s u b c u l t u r e ( l - 9 ) Total number on b a s a l m e d i u m + 3 ~ M T I B A b of p l a n t s per 2. 3. 4. 5. 6. 7. 8. 9. explant

2r4-D

7 3 2

_c 4 0

2,4,5-T

13 5 22 6 22 6 22 6 452 ii 7 ii 7 ii 7 ii 7 195 195 19 5 19.5 31.3 39.1 39.1

(1)

aBasal

medium

+ 4.5%

medium

+ 2% s u c r o s e not

_ .

-

sucrose

been

.

_ 0 1

_ 0 -

1

0

-

-

0

0

4

16

.

1

(1)

bBasal

have

.

I0

T o t a l n u m b e r of p l a n t s per s u b c u l t u r e :

CCultures

9 4 2 2 4 9

-

.

.

.

.

7 7 23 (1)

12

(1)

(1)

.

.

(1)

.

.

3 5 2

2 2 2

(Z) (i)

. .

0 4 5

5 0 13

0 0 7

0 1 1

0 0 2

8

3

0

1

5 5 40

41

28

i0

4

2

8

3

4

17

117

2 0 0 . .

(1)

0 0 0 . .

+ 1% a g a r

+ 1% a g a r

subcultured

(1)

on T I B A

0 0 0

7 7 4

. .

(1) (i)

128

Fig. i. Barley callus line (4.36/3) cultured on MS basal medium with I0 ~M 2,4-D + i0 ~M IAA + 3% sucrose + 0.8 % agar. The 8th subculture. Bar=l cm Fig. 2. Rhizogenic tissue culture of barley (4.8/3) grown for a long time on MS mediun + 3 ~4 TIBA + 2% sucrose + 1% agar. The 14th subculture. Fig. 3. Shoot development in the 8th subculture of barley callus (4.36/3) transferred successively onto MS mediua + 3 llM TIBA + 2% sucrose + 1% agar. Four-week-old culture. Fig. 4. Advanced growth of shoots from callus with TIBA added to the m e d i ~ (4.36/3) after transfer on the MS + 5.7 ~M IAA + 3% sucrose + 1% ager. Two-wt~_k-old culture.

Fig. 5. Complete barley plants, generated in callus culture originating from a mature embryo (4.36/3), after 4 weeks in soil. Table 4. Percentages of cultures r e g e n e r a t i n g shoots in barley callus (4.36/3) subcultured on m e d i a supplemented with TIBA or with 2,4,5-T. In the ist subculture all cultures were subcultured on TIBA, in next parts of the same cultures were transferred on TIBA and 2,4,5-T m e d i a parallel. Subculture

T I BA a Cultures ~ o o t s per total number of cultures

No. i. 2. 3. 4. 5.

15/46 6/17 3/15 3/11 1/16

32.6 35.3 20.0 27.3 6.3

~ Number of regenerated plants

b

C u l t u r e s w i t h shoots per total number of cultures %

4 7

-

28 I0 4 2

1/15 1/18 2/26 0/20

Number of regenerated plants

-

-

6.7 5.6 7.4 0

2 1 5 0

aBasal m e d i u m + 2% sucrose + 1% agar + 3 ~ M TIBA bBasal m a d i u m + 4.5% sucrose + 1% agar + 19.5 ~M 2,4,5-T 1975} S a a l b a c h and Koblitz, 197~; Dale and Deambrogio, 1979, 1980). We used the same procedure. In c o m p a r i s o n w i t h other r e g e n e r a t i on m e d i a used, the m e d i u m with 3 itM TIBA enabled shoot f o r m a t i o n m u c h more frequently. Furthermore, shoot regenerationw connected m o s t l y with green spots, existed in rhizogenic cultures. H o w e v e r , s o m e cultures w i t h the same m o r p h o g e n i c c h a r a c t e r i s t i c s were apparen-

tly not capable of shoot formation. E x p e r i m e n t s r e p o r t e d p r e v i o u s l y (Cheng and Smith, 1975; Scheunert et al. 1977; Kartel and Maneshina, 1978; Dale and Deambrogio, 1979) showed that r e g e n e r a t i v e c a p a c i t y of barley callus d e p e n d e d on genotype and that it d e c r e a s e d during succesive subcultures. This is also being confirmed by our recent results. We would like to emphasize, however,

129 the effect of TIBA (3 DM) on the increased expression of shoot formation. Similarly in rice embryogenic callus plant r e g e n e r a t i o n was also enhanced when callus was subcultured on a medium containing TIBA as opposed to IAA (Nabors, 1982). In both cases r e g e n e r a t i o n in callus seems to require low auxin level which could be achieved by an auxin transport inhibitor like TIBA. How rhizogenic cultures could grow during many subcultures on the m e d i u m with added 3 ~M TIBA and without auxin is difficult to e x p l a i ~ Tissue h a b i t u a t i o n by high auxin concentrations in primary cultures might be considered. A big difference in the capacity of shoot formation (from four to forty) among mesocotyl explants of the same genotype is also difficult tc explail . Cytological analyses of r e g e n e r a t e d barley plants and callus tissue (Genotype 4.36/3) were partly reported (Jelaska et al. 1981~ Pape~ et al. 1983). They showed m o s a i c i s m of chromosome number (2n=14, 15, 16) and other deviations in chromosome behaviour. However, the initial material from which tissue culture was induced and established exibited the same types of chromosome changes (different chromosome number, m o s a i c i s m and anaphase bridges with laggings). ACKNOWLEDGEMENTS We thank Mr. J. Kovadevi~ (Agricultural Institute-Osijek) for supplying us with barley seeds used in this work, and Prof. W.J. Libby (Berkeley) and Mrs. Mia Pervan-Plavec for their help in preparing the English text. This research was supported by the Science Research Council of SR Croatia (SIZ-IV).

REFERENCES Cheng TV, Smith HH (1975) Planta (Berl) 123: 307-310 Dale PJ, Deambrogio E (1979) Z Pflanzenphysiol 9 4 : 6 5 - 7 7 Dale PJ, Deambrogio E (1980) Cereal Research Communication 8 / 2 : 4 1 7 - 4 2 3 Jelaska S, Pape~ D, Rengel Z, Garaj-Vrhovec V, Cesar V (1981) 2nd Yugoslav Genetic Conference, Abstr. Book: 86 Kartel NA, Maneshina TV (1978) Plant Physiol (Moscow) 2 5 : 2 8 3 - 2 8 7 Murashige T, Skoog F (1962) Physiol Plant 15: 473-497 Nabors MW (1982) Progress Report, Tissue Culture for Crops Project, Dept Botany and Plant Pathology, Colorado State University, Fort Collins Orton TJ (197~) Envir Exp Botany 1 9 : 3 1 9 - 3 3 5 Pape~ D, Garaj-Vrhovec V, Jelaska S, KolevskaPletikapi~ B (1983) Kew Chromosome Conference II. George Allen & Unwin, London, pp 155-163 Pental D, Gunckel JE (1977) In: Sharp WR, Larsen PO, Paddock EF, Raghavan V (Eds) Plant CeLl and Tissue Culture, Ohio State University, pp 633-709 Rice TB, Reid RK. Gordon PN (1978) In: Hughes KW, HenCe R r Constantin M (Eds) Pro-, pagation of Higher Plants through Tissue Culture, A B r i d g e between Research and Application, US Dept Commerce NTIS, pp 262-277 Saalbach G, Koblitz H (1978) Plant Sci Lett 13:165-169 Scheunert EU, Shamina ZB, Koblitz H (1977) Plant Sci Lett 1 0 : 3 1 3 - 3 1 8