Plant Cell Reports
P l a n t Cell R e p o r t s (1992) 1 1 : 3 9 - 4 3
9 Springer-Verlag 1992
Callus formation and plant regeneration in standard and microexplants from seedlings of barley (Hordeum vulgare L.) Tiirid Becher, Gudrun Haberland, and Hans-Ulrich Koop L a b o r a t o r y for P l a n t Cell Biology a n d Cell C u l t u r e Botanical Institute, U n i v e r s i t y o f M u n i c h , M e n z i n g e r Strasse 67, W-8000 M f i n c h e n 19, F R G Received A u g u s t 13, 1991/Revised version received O c t o b e r 12, 1991 - C o m m u n i c a t e d by H. L6rz
Abstract. Callus was induced in standard (1 mm) leaf base explants and in cross sections (microexplants) through the seedling axis from seedlings of Hordeum vulgare L.. Reduced callus formation was observed with increasing distance from the leaf base, and explants from the first and second leaves gave the best response. In serial hand sections of the seedling axis frequency of callus formation decreased from 100% in the apical region to 5 % in the basal region. Callus formed from all tissues outside the central vascular elements, except for the coleoptile and the scutellum. Plants were regenerated from callus induced from both types of explants.
Materials and m e t h o d s
Donor plants. Seeds of cultivar "lgri" were kindly provided by the Inst. of resistance genetics; Griinbach, cultivars "Rumba", "Trixi" and "Steffi" were supplied by Saatzucht Ackermann, Irlbach. Seeds were imbibed with water overnight, husks were removed and sterilization was performed by a 1 h incubation in 15% H202, containing 0,05% Tween 80. Seeds were directly plated on 15 ml MS medium (without growth regulators, 2% sucrose, 0.7% agar) in tissue culture tubes and germinated at 23~
in the light (16 h, 4000 Ix)). After four to five days
seedlings were transferred to 120 ml of the same medium, but solidified with 0,2% Gelrite, in food jars of 720 ml total volume supplied with a foam plug for gas exchange.
Erplant preparation. Explants were prepared from 10 to 12 day old seedlings. The third leaf was visible at this stage and the fourth leaf had
Introduction
a size of up to 20 mm. For leaf base segments, the leaves were trimmed down to approximately 2 cm, removed from the seedling axis,
Whereas many methods are available for biotechnological modification of plants at the cellular level, their application for crop improvement is often hampered by the difficulty to regenerate whole plants from the cellular systems required. In barley, the recent reports on regeneration of fertile plants from protoplasts (Yan et al. 1990, J~ihne et al. 1991) can be regarded as a significant contribution to this problem. However, the methods used have a number of disadvantages. Regeneration relies on the availability of embryogenic suspensions and regeneration has only been achieved with low efficiency and with a limited number of cultivars. Therefore, it is still of interest to develop alternative regeneration systems in barley. In our research programme we are aiming at the identification of tissues or cells which are competent of regenerating whole plants. Our study focusses on seedlings, since such tissue provides a readily accessible donor material for explants. Here we report on callus induction and plant regeneration from standard leaf base explants and from microexplants derived from the seedling axis. Offprint requests to." H . - U . K o o p
freed of axillary buds and cut into 1 m m perpendicular sections using a scalpel. For seedling axis microexplants, the seedling was removed from the grain and the leaves and roots were cut away close to the seedling axis. The axis was then mounted in a sterile pith block and serial hand sections of the region between the dorsal extension of the scutellum and the base of the second leaf (Fig. 1) were prepared with a razor blade and immediately transferred to callus induction medium.
Evaluation of culture response in microexplants. The number of sections recovered from each seedling axis varied between eight and fifteen. Thickness was between 0.l and 0,2 mm, representing about 10 to 20 cell layers. The relative position of a particular section whithin its axis was calculated by assigning the values zero and one for the most basal and most apical sections, respectively.
Callus induction. Standard- and microexplants were cultured in 24Multiwell dishes on 1 ml aliquots of MS medium, solidified with 0,2% Gelrite and containing 2%
sucrose and 2 mg/L 2,4-D. Where
appropriate, the induction medium was modified to contain various carbohydrates and auxins. Culture conditions were the same as for the seedlings; transfer to fresh medium was performed at four week
40 10o
~
coieoptile leaf 2nd leaf
8O
1st
leaf
3rd
6O
OQ
~o
9
% callus induction (27 d)
40
!
leaf
""
0~. e ~
ellum inal root grain
20
0 1
root
2
3
4
5
6
segment number
m leaf1 ~leaf 2 ~leaf 3 ~leaf 4 Fig.1. Schematical representation o f a longitudinal section through a
Fig.2. Influence of the explant position in the seedling on callus
barley seedling at the developmental stage used for preparing leaf base
induction frequencies in leaf base segments o f barley (ev. "Igri").
segments
Segment numbers represent consecutive segments starting from the seedling axis (segment 1).
(standard
explants)
and
seedling axis
cross
sections
(microexplants). The relative positions o f sections in the seedling axis are indicated by the scale. intervals. Callus formation was monitored microscopically, and the diameter o f formed callus was measured. Callus of more than 1 mm diameter was scored as successful induction.
Plant regeneration. Shoots or somatic embryos which formed either on callus induction medium or regenerated a~er transfer o f compact callus to regeneration medium (MS medium, 0,2% Gelrite, 2% sucrose, 0.05
2. Gelling agents. When comparing callus induction on agar (0.7%, Difco Bacto), agarose (0.7%, BRL, low melting point) and Gelrite (0~2%), the highest callus induction rates were found on Gelrite. The frequencies, determined after 24 days of culture (total of eight seedlings), were 92% (Gelrite), 72% (agarose) and 66% (agar). Thus, for all further experiments Gelrite was used.
mg/L 2,4-D, with 0,2 to 1 mg/L kinetin or 0.5 to 1 mg/L BAP), were transferred to MS medium without growth regulators. A limited number o f the regenerated plants was established in soil, where they developed into normal, fertile plants.
Histological analysis. Fixation, postfixation, staining, dehydration and embedding followed standard procedures as described by Amman et al. (1986). Mierotome (Pyramitome, LKB) sections o f 4 ~
thickness
were cut using glass knifes. The sections were mounted on microscope slides and inspected using bright field, phase contrast or DIC-optics.
Results
Standard explants (leaf base) 1. Position of the explant in the donor seedling. The frequency of callus formation in leaf base explants was strongly dependent on the position in the seedling (Fig. 2). The highest response was found in leaves one and two. A pronounced gradient existed within each leaf: the segments closest to the seedling axis showed much higher callus formation frequencies than the more distant ones. Thus, for further analysis the first segments from the first two or three leaves were used.
3. Carbohydrates. When comparing callus induction frequencies in the presence of different carbohydrates in the medium (2% each), highest induction rates were found with sucrose, followed by maltose, glucose, cellobiose and fructose (Fig. 3). Browning and necrosis occured in the presence of cellobiose and fructose after prolonged culture. Supplying sugar alcohols (mannitol or sorbitol) in addition to 2% sucrose proved inhibitory at all concentrations tested (1.5% to 6%). Callus formation was observed in only 30% (mannitol) and 25% (sorbitol) of the explants at the highest concentration tested. 4. Auxins. Different auxins were investigated with respect to their callus induction capacity in leaf base explants (Fig. 4). Callus formation was observed with all the auxins tested. The highest response was observed with NAA, followed by 2,4-D, dicamba, pCPA and IAA. 5. Cultivars. From the four cultivars investigated, three gave almost identical callus induction frequencies. Response was reduced in "Steffi", mainly due to pronounced browning and cell death after prolonged culture.
41 the compact calli at a concentration of 0,1 mg/L, from 8% at 0,2 mg/L, from 28% at 0,5 mg/L and from 24% at 1 mg/L, respectively. Callus induction frequencies varied by a factor of two between the different leaves, from which the explants were made (Fig. 2). Regeneration capacity of these lines also differed by a factor of two. 8,9% of the explants from the first leaf gave regenerated plants, we recovered plants from 5,2% of the explants from the second leaf, 4,2% from the third and 4,8% from the fourth leaf, respectively.
% callus induction 111
60
.......................................................................
4 0
.........
...................................................
Microexplants (seedling axis) o
4 weeks
8 weeks
duration of culture m
sucrose
~
maltose
cellobiose
~
fructose
~
glucose
Fig.3. Influence of the carbon source on callus induction frequencies in leaf base segments of barley (cv. "Igri"). Carbohydrate concentration was 2%, no callus formation was observed with mannose, ribose, or xylose. 7O
% callus induction
40
1. Survival of the microexplant tissues. The tissues included in the hand sections prepared from the seedling axis survived the sectioning process. Thus, microexplants represent an alternative explant type for the analysis of callus induction and regeneration in barley. 2. Position of the microexplant in the donor seedling. Callus of 0.5 to 1 mm diameter could be observed as early as three to four days of culture. After one week of culture induction frequencies approaching 80% were found in explants derived from the apical region of the axis (Fig. 5). Frequencies varied dramatically with respect to the position of the explant in the donor seedling axis (Fig. 5). Explants from the relative positions 0.1 to 0.3 (compare: methods section) formed callus at very low frequencies or not at all. The sections turned brown within the first two to three weeks of culture, and necrosis was frequently observed. Induction frequencies gradually increase with increasing distance from the basal
30
% callus induction lOO
20
10
0
80
IAA
pCPA
Dicarnba
auxin 2 weeks
2,4-D
NAA
(2,0 mg/I) ~
60
8 weeks
Fig.4. Influence of various auxins on callus induction frequencies in
40
leaf base segments of barley (cv. "Igri"). The induction frequencies are given after 2 weeks and 8 weeks of culture. 20
6. Plant regeneration.
Callus formation was first observed at the wound edges of the segments. Initially, transparent soft callus appeared, later we observed compact yellowish callus in some of the explants. This callus was transferred to a number of regeneration media. From a total of 30 different compact callus lines plated only two (7%) regenerated plants on media containing BAP (0.5 mg/L, 1 rag/L). Regeneration efficiency was higher with kinetin; plants were regenerated from 20 % of
0 0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
relative position m
1 week
~
6 weeks
Fig.5. Influence of the relative positions of hand sections derived from the axis of seedlings of barley (cv. "Igri") on callus induction frequencies after 1 week and 6 weeks of culture.
42 end of the axis. Nearly 100% of the explants, derived from the relative positions 0,8 to 1,0, responded with callus formation.
3. Influence of preculture conditions and sucrose concentration. We analyzed a number of preculture conditions with respect to their possible influence on viability and callus induction frequency in microexplants. No difference was observed due to each of the following conditions: age of the seedlings 6 days or 12 days, etiolated or non-etiolated seedlings, pretreatment with 2 mg/L 2,4-D during the first half of the 12 d preculture period or preculture without 2,4-D. Sucrose concentration also did not drastically influence callus formation frequencies. When results were calculated from all microexplants, the frequencies were determined to 35% (0.125% sucrose), 45% (0~25% sucrose), 53% (0,5% sucrose), 55% (1% sucrose), 56% (2% sucrose), 65% (4% sucrose) and 32% (8% sucrose) respectively. It was observed, however, that no compact callus and no shoot or embryo formation occured from callus induced at sucrose concentrations below 1%.
4. Regeneration. Regeneration of shoots or embryos was only observed from callus induced in sections from the upper parts of the seedling axis (relative position 0,8 to 1,0). As in 1 mm leaf base explants soft and transparent callus developed initially also in seedling axis microexplants. Compact callus developed after four to six weeks of culture. Shoot and embryo formation was also observed.
5. Site of callus formation. Histological analyis was performed with fresh explants in order to identify the tissues from which callus induction occurs. Since regeneration was observed to occur only from the upper parts of the seedling axis special emphasis was placed on the analysis of this region. A typical cross section is shown in Fig. 6. Callus formation occurred from all tissues outside the central vascular elements except for the coleoptile and, in sections derived from more basal regions of the seedling axis, the scutellum.
Discussion Regeneration in barley as well as in other cereals has been observed from various explants derived from different tissues. The main sources for the induction of morphogenic or embryogenic callus are immature or mature embryos, immature inflorescenees, apical meristems and leaf base (for a review on barley see Dunwell 1986). Clearly, the most easily available donor material in barley are seedlings, since mature plants and specialized growth environments are not required. We have therefore focussed our investigations on seedlingderived explants.
Leaf base (standard explants). The suitability of the leaf base as explant in barley was reported earlier (Jelaska et al. 1984, Mohanty and Ghosh 1988). Our results confirm the presence of a pronounced gradient of morphogenic capacity in leaves of cereals such as rice (Wemicke et al. 1981), Sorghum (Wernicke and Brettel 1980, 1982) and wheat (Ahuja et al. 1982, Zamora and Scott 1983, Wemicke and Milkowits 1984). In barley, different culture response depending on the explant position in the leaf was also described by Mohanty and Ghosh (1988). In contrast to their observations that callus could only be induced in explants from the third or the subsequent leaves, we observed callus induction and plant regeneration from all four leaves investigated. Such a difference might be due to the different cultivar ("Karan 92") used in their studies. Among the different leaves, induction frequencies varied by a factor of two (Fig. 2). A similar result was observed for the frequency of plant regeneration: e.g. 8.9% in callus derived from the first and 4.8% in callus derived from the fourth leaf, respectively. This might indicate, that although callus induction frequencies are higher in the first and second leaf, the regeneration capacity of the callus is not different. Therefore, more callus, but not callus with superior regeneration capacity can be produced from the first and second leaf. The results obtained when studying the influence of various experimental parameters on callus induction rates, i.e. gelling agents, carbohydrates, auxins, are in accordance with the general experience in other cereals. Since we have focussed our investigations on callus formation and not plant regeneration, it cannot be excluded that some of the parameters determined might be less suitable for achieving optimum regeneration frequencies.
Microexplants (seedling axis). Microexplants were used for the first time in cereals in the course of these investigations. A number of reasons might make microexplants superior to standard explants. Firstly, the amount of callus recovered is higher, secondly, the type of response might be different, thirdly, microexplants allow identification of responding tissues at higher resolution, and, in addition, microexplants might allow cell biological techniques to be used, such as microinjection or particle bombardment, which cannot be used when applied to standard explants. The amount of callus recovered from a series of hand sections through a seedling axis is higher than the amount recovered when using the axis as a single explant. A similar observation was reported in white clover (Bond and Webb 1989) and poplar (Lee-Stadelmann et al. 1989). "Thin cell layers" (Tran Than Van 1973) are well known to represent explants leading to unique morphogenetic responses (e.g. tobacco: Tran Than Van and Thrin 1986, rapeseed: Klimaszewska and Keller 1985, sugar beet:
43
Fig.6. Microtome cross section through the apical region of the
Amman M, Wanner G, Zenk MH (1986) Planta 167:310-320
seedling axis of a 12 day old seedling of barley (cv. "Igri").
Bond JE, Webb KJ (1989) Plant Sci. 61:119-125
Detrez et al. 1988, sunflower: Pelissier et al. 1990). Although the handsections used in these investigations do not represent typical "thin cell layers", it cannot be excluded that under conditions which are unknown as yet, qualitatively different responses from those of standard explants might be experienced. Microexplants allow identification of responding tissues at higher degrees of resolution. These investigations are an initial attempt at exploiting this approach for seedlings of barley. Sections from the apical part of the seedling axis (Fig. 5) showed the highest callus induction frequencies. Callus formation occured from all the tissues outside the central vascular elements, excluding however, the coleoptile and the scutellum. In contrast, the scutellum and coleoptile are known to be the responding tissues in immature embryo explants in a variety of Poaceae (Vasil and Vasil 1982, Lu and Vasil 1982, Lu et al. 1983, Ozias-Akins and Vasil 1982, 1983). Our future studies will concentrate on the plant regeneration capabilities of seedling axis-derived sections. We eventually hope to establish systems which are compentent for both transformation and regeneration.
Detrez C, Tetu T, Sangwan RS, Sangwan-Noreel BS (1988) J. Exp.
Acknowledgements The help of Dr. G. Wanner with the histological analysis is gratefully appreciated. The work was funded by BMFT (Bundesministerium fiir Forschung und Technologie), grant 0319398A.
Bot. 39:917-926 Dunwell JM (1986) Barley, In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of plant cell culture, vol IV, Macmillan Publishing Company, New York, pp 339-369 J~ihne A, Lazzefi PA, L6rz H (1991) Plant Cell Rep. 10:1-6 Jelaska S, Rengel Z, Cesar V (1984) Plant Cell Rep. 3:125-129 Klimaszewska K, Keller WS (1985) Plant Cell Tissue Organ Culture 4: 183-197 Lee-Stadelman OY, Loo SW, Hackett WP, Read PE (1989) Plant Sci. 61 : 263-272 Lu CY, Vasil IK (1982) Amer. J. Bot. 69:77-81 Lu CY, Vasil V, Vasil IlK (1983) Theor. Appl. Genet. 66:265-289 Mohanty BD, Ghosh PD (1988) Ann. Bot. 61:551-555 Ozias-Akins P, Vasil IK (1982) Protoplasma 110:95-105 Ozias-Akins P, Vasil IK (1983) Protoplasma 117:40-44 P61essier B, Bouchefra O, P@in R, Freyssinet G (1990) Plant Cell Rep. 9:47-50 Tran Than Van M (1973) Planta 115:87-92 Tran Than Van M, Thrln TH (1986)
In: Evans DA, Sharp WR,
Ammirato PV, Yamada Y (eds) Handbook of plant cell culture, vol IV, Macmillan Publishing Company, New York, pp 316-335 Vasil V, Vasil IK (1982) Bot. Gaz. 143:454-465 Wernicke W, Brettell R (1980) Nature 287:138-139 Wernicke W, Brettell R (1982) Protoplasma 111 : 19-27 Wernicke W, Milkovits L (1984) J. Plant Physiol. 115:49-58 Wernicke W, Brettell R, Wakizuka T, Potrykus I (1981) Zeitschr.
References Ahuja PS, Pental D, Cocking EC (1982) Zeltschr. Pflanzenzficht. 89: 139-144
Pflanzenphysiol. 103:361-365 Yan Q, Zhang X, Shi J, Li J (1990) Kexue Tongbao 35: in press Zamora AB, Scott KJ (1983) Plant Sci. Lett. 29: 29:183-189
P l a n t Cell R e p o r t s (1992) 1 1 : 3 9 - 4 3
9 Springer-Verlag 1992
Callus formation and plant regeneration in standard and microexplants from seedlings of barley (Hordeum vulgare L.) Tiirid Becher, Gudrun Haberland, and Hans-Ulrich Koop L a b o r a t o r y for P l a n t Cell Biology a n d Cell C u l t u r e Botanical Institute, U n i v e r s i t y o f M u n i c h , M e n z i n g e r Strasse 67, W-8000 M f i n c h e n 19, F R G Received A u g u s t 13, 1991/Revised version received O c t o b e r 12, 1991 - C o m m u n i c a t e d by H. L6rz
Abstract. Callus was induced in standard (1 mm) leaf base explants and in cross sections (microexplants) through the seedling axis from seedlings of Hordeum vulgare L.. Reduced callus formation was observed with increasing distance from the leaf base, and explants from the first and second leaves gave the best response. In serial hand sections of the seedling axis frequency of callus formation decreased from 100% in the apical region to 5 % in the basal region. Callus formed from all tissues outside the central vascular elements, except for the coleoptile and the scutellum. Plants were regenerated from callus induced from both types of explants.
Materials and m e t h o d s
Donor plants. Seeds of cultivar "lgri" were kindly provided by the Inst. of resistance genetics; Griinbach, cultivars "Rumba", "Trixi" and "Steffi" were supplied by Saatzucht Ackermann, Irlbach. Seeds were imbibed with water overnight, husks were removed and sterilization was performed by a 1 h incubation in 15% H202, containing 0,05% Tween 80. Seeds were directly plated on 15 ml MS medium (without growth regulators, 2% sucrose, 0.7% agar) in tissue culture tubes and germinated at 23~
in the light (16 h, 4000 Ix)). After four to five days
seedlings were transferred to 120 ml of the same medium, but solidified with 0,2% Gelrite, in food jars of 720 ml total volume supplied with a foam plug for gas exchange.
Erplant preparation. Explants were prepared from 10 to 12 day old seedlings. The third leaf was visible at this stage and the fourth leaf had
Introduction
a size of up to 20 mm. For leaf base segments, the leaves were trimmed down to approximately 2 cm, removed from the seedling axis,
Whereas many methods are available for biotechnological modification of plants at the cellular level, their application for crop improvement is often hampered by the difficulty to regenerate whole plants from the cellular systems required. In barley, the recent reports on regeneration of fertile plants from protoplasts (Yan et al. 1990, J~ihne et al. 1991) can be regarded as a significant contribution to this problem. However, the methods used have a number of disadvantages. Regeneration relies on the availability of embryogenic suspensions and regeneration has only been achieved with low efficiency and with a limited number of cultivars. Therefore, it is still of interest to develop alternative regeneration systems in barley. In our research programme we are aiming at the identification of tissues or cells which are competent of regenerating whole plants. Our study focusses on seedlings, since such tissue provides a readily accessible donor material for explants. Here we report on callus induction and plant regeneration from standard leaf base explants and from microexplants derived from the seedling axis. Offprint requests to." H . - U . K o o p
freed of axillary buds and cut into 1 m m perpendicular sections using a scalpel. For seedling axis microexplants, the seedling was removed from the grain and the leaves and roots were cut away close to the seedling axis. The axis was then mounted in a sterile pith block and serial hand sections of the region between the dorsal extension of the scutellum and the base of the second leaf (Fig. 1) were prepared with a razor blade and immediately transferred to callus induction medium.
Evaluation of culture response in microexplants. The number of sections recovered from each seedling axis varied between eight and fifteen. Thickness was between 0.l and 0,2 mm, representing about 10 to 20 cell layers. The relative position of a particular section whithin its axis was calculated by assigning the values zero and one for the most basal and most apical sections, respectively.
Callus induction. Standard- and microexplants were cultured in 24Multiwell dishes on 1 ml aliquots of MS medium, solidified with 0,2% Gelrite and containing 2%
sucrose and 2 mg/L 2,4-D. Where
appropriate, the induction medium was modified to contain various carbohydrates and auxins. Culture conditions were the same as for the seedlings; transfer to fresh medium was performed at four week
40 10o
~
coieoptile leaf 2nd leaf
8O
1st
leaf
3rd
6O
OQ
~o
9
% callus induction (27 d)
40
!
leaf
""
0~. e ~
ellum inal root grain
20
0 1
root
2
3
4
5
6
segment number
m leaf1 ~leaf 2 ~leaf 3 ~leaf 4 Fig.1. Schematical representation o f a longitudinal section through a
Fig.2. Influence of the explant position in the seedling on callus
barley seedling at the developmental stage used for preparing leaf base
induction frequencies in leaf base segments o f barley (ev. "Igri").
segments
Segment numbers represent consecutive segments starting from the seedling axis (segment 1).
(standard
explants)
and
seedling axis
cross
sections
(microexplants). The relative positions o f sections in the seedling axis are indicated by the scale. intervals. Callus formation was monitored microscopically, and the diameter o f formed callus was measured. Callus of more than 1 mm diameter was scored as successful induction.
Plant regeneration. Shoots or somatic embryos which formed either on callus induction medium or regenerated a~er transfer o f compact callus to regeneration medium (MS medium, 0,2% Gelrite, 2% sucrose, 0.05
2. Gelling agents. When comparing callus induction on agar (0.7%, Difco Bacto), agarose (0.7%, BRL, low melting point) and Gelrite (0~2%), the highest callus induction rates were found on Gelrite. The frequencies, determined after 24 days of culture (total of eight seedlings), were 92% (Gelrite), 72% (agarose) and 66% (agar). Thus, for all further experiments Gelrite was used.
mg/L 2,4-D, with 0,2 to 1 mg/L kinetin or 0.5 to 1 mg/L BAP), were transferred to MS medium without growth regulators. A limited number o f the regenerated plants was established in soil, where they developed into normal, fertile plants.
Histological analysis. Fixation, postfixation, staining, dehydration and embedding followed standard procedures as described by Amman et al. (1986). Mierotome (Pyramitome, LKB) sections o f 4 ~
thickness
were cut using glass knifes. The sections were mounted on microscope slides and inspected using bright field, phase contrast or DIC-optics.
Results
Standard explants (leaf base) 1. Position of the explant in the donor seedling. The frequency of callus formation in leaf base explants was strongly dependent on the position in the seedling (Fig. 2). The highest response was found in leaves one and two. A pronounced gradient existed within each leaf: the segments closest to the seedling axis showed much higher callus formation frequencies than the more distant ones. Thus, for further analysis the first segments from the first two or three leaves were used.
3. Carbohydrates. When comparing callus induction frequencies in the presence of different carbohydrates in the medium (2% each), highest induction rates were found with sucrose, followed by maltose, glucose, cellobiose and fructose (Fig. 3). Browning and necrosis occured in the presence of cellobiose and fructose after prolonged culture. Supplying sugar alcohols (mannitol or sorbitol) in addition to 2% sucrose proved inhibitory at all concentrations tested (1.5% to 6%). Callus formation was observed in only 30% (mannitol) and 25% (sorbitol) of the explants at the highest concentration tested. 4. Auxins. Different auxins were investigated with respect to their callus induction capacity in leaf base explants (Fig. 4). Callus formation was observed with all the auxins tested. The highest response was observed with NAA, followed by 2,4-D, dicamba, pCPA and IAA. 5. Cultivars. From the four cultivars investigated, three gave almost identical callus induction frequencies. Response was reduced in "Steffi", mainly due to pronounced browning and cell death after prolonged culture.
41 the compact calli at a concentration of 0,1 mg/L, from 8% at 0,2 mg/L, from 28% at 0,5 mg/L and from 24% at 1 mg/L, respectively. Callus induction frequencies varied by a factor of two between the different leaves, from which the explants were made (Fig. 2). Regeneration capacity of these lines also differed by a factor of two. 8,9% of the explants from the first leaf gave regenerated plants, we recovered plants from 5,2% of the explants from the second leaf, 4,2% from the third and 4,8% from the fourth leaf, respectively.
% callus induction 111
60
.......................................................................
4 0
.........
...................................................
Microexplants (seedling axis) o
4 weeks
8 weeks
duration of culture m
sucrose
~
maltose
cellobiose
~
fructose
~
glucose
Fig.3. Influence of the carbon source on callus induction frequencies in leaf base segments of barley (cv. "Igri"). Carbohydrate concentration was 2%, no callus formation was observed with mannose, ribose, or xylose. 7O
% callus induction
40
1. Survival of the microexplant tissues. The tissues included in the hand sections prepared from the seedling axis survived the sectioning process. Thus, microexplants represent an alternative explant type for the analysis of callus induction and regeneration in barley. 2. Position of the microexplant in the donor seedling. Callus of 0.5 to 1 mm diameter could be observed as early as three to four days of culture. After one week of culture induction frequencies approaching 80% were found in explants derived from the apical region of the axis (Fig. 5). Frequencies varied dramatically with respect to the position of the explant in the donor seedling axis (Fig. 5). Explants from the relative positions 0.1 to 0.3 (compare: methods section) formed callus at very low frequencies or not at all. The sections turned brown within the first two to three weeks of culture, and necrosis was frequently observed. Induction frequencies gradually increase with increasing distance from the basal
30
% callus induction lOO
20
10
0
80
IAA
pCPA
Dicarnba
auxin 2 weeks
2,4-D
NAA
(2,0 mg/I) ~
60
8 weeks
Fig.4. Influence of various auxins on callus induction frequencies in
40
leaf base segments of barley (cv. "Igri"). The induction frequencies are given after 2 weeks and 8 weeks of culture. 20
6. Plant regeneration.
Callus formation was first observed at the wound edges of the segments. Initially, transparent soft callus appeared, later we observed compact yellowish callus in some of the explants. This callus was transferred to a number of regeneration media. From a total of 30 different compact callus lines plated only two (7%) regenerated plants on media containing BAP (0.5 mg/L, 1 rag/L). Regeneration efficiency was higher with kinetin; plants were regenerated from 20 % of
0 0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
relative position m
1 week
~
6 weeks
Fig.5. Influence of the relative positions of hand sections derived from the axis of seedlings of barley (cv. "Igri") on callus induction frequencies after 1 week and 6 weeks of culture.
42 end of the axis. Nearly 100% of the explants, derived from the relative positions 0,8 to 1,0, responded with callus formation.
3. Influence of preculture conditions and sucrose concentration. We analyzed a number of preculture conditions with respect to their possible influence on viability and callus induction frequency in microexplants. No difference was observed due to each of the following conditions: age of the seedlings 6 days or 12 days, etiolated or non-etiolated seedlings, pretreatment with 2 mg/L 2,4-D during the first half of the 12 d preculture period or preculture without 2,4-D. Sucrose concentration also did not drastically influence callus formation frequencies. When results were calculated from all microexplants, the frequencies were determined to 35% (0.125% sucrose), 45% (0~25% sucrose), 53% (0,5% sucrose), 55% (1% sucrose), 56% (2% sucrose), 65% (4% sucrose) and 32% (8% sucrose) respectively. It was observed, however, that no compact callus and no shoot or embryo formation occured from callus induced at sucrose concentrations below 1%.
4. Regeneration. Regeneration of shoots or embryos was only observed from callus induced in sections from the upper parts of the seedling axis (relative position 0,8 to 1,0). As in 1 mm leaf base explants soft and transparent callus developed initially also in seedling axis microexplants. Compact callus developed after four to six weeks of culture. Shoot and embryo formation was also observed.
5. Site of callus formation. Histological analyis was performed with fresh explants in order to identify the tissues from which callus induction occurs. Since regeneration was observed to occur only from the upper parts of the seedling axis special emphasis was placed on the analysis of this region. A typical cross section is shown in Fig. 6. Callus formation occurred from all tissues outside the central vascular elements except for the coleoptile and, in sections derived from more basal regions of the seedling axis, the scutellum.
Discussion Regeneration in barley as well as in other cereals has been observed from various explants derived from different tissues. The main sources for the induction of morphogenic or embryogenic callus are immature or mature embryos, immature inflorescenees, apical meristems and leaf base (for a review on barley see Dunwell 1986). Clearly, the most easily available donor material in barley are seedlings, since mature plants and specialized growth environments are not required. We have therefore focussed our investigations on seedlingderived explants.
Leaf base (standard explants). The suitability of the leaf base as explant in barley was reported earlier (Jelaska et al. 1984, Mohanty and Ghosh 1988). Our results confirm the presence of a pronounced gradient of morphogenic capacity in leaves of cereals such as rice (Wemicke et al. 1981), Sorghum (Wernicke and Brettel 1980, 1982) and wheat (Ahuja et al. 1982, Zamora and Scott 1983, Wemicke and Milkowits 1984). In barley, different culture response depending on the explant position in the leaf was also described by Mohanty and Ghosh (1988). In contrast to their observations that callus could only be induced in explants from the third or the subsequent leaves, we observed callus induction and plant regeneration from all four leaves investigated. Such a difference might be due to the different cultivar ("Karan 92") used in their studies. Among the different leaves, induction frequencies varied by a factor of two (Fig. 2). A similar result was observed for the frequency of plant regeneration: e.g. 8.9% in callus derived from the first and 4.8% in callus derived from the fourth leaf, respectively. This might indicate, that although callus induction frequencies are higher in the first and second leaf, the regeneration capacity of the callus is not different. Therefore, more callus, but not callus with superior regeneration capacity can be produced from the first and second leaf. The results obtained when studying the influence of various experimental parameters on callus induction rates, i.e. gelling agents, carbohydrates, auxins, are in accordance with the general experience in other cereals. Since we have focussed our investigations on callus formation and not plant regeneration, it cannot be excluded that some of the parameters determined might be less suitable for achieving optimum regeneration frequencies.
Microexplants (seedling axis). Microexplants were used for the first time in cereals in the course of these investigations. A number of reasons might make microexplants superior to standard explants. Firstly, the amount of callus recovered is higher, secondly, the type of response might be different, thirdly, microexplants allow identification of responding tissues at higher resolution, and, in addition, microexplants might allow cell biological techniques to be used, such as microinjection or particle bombardment, which cannot be used when applied to standard explants. The amount of callus recovered from a series of hand sections through a seedling axis is higher than the amount recovered when using the axis as a single explant. A similar observation was reported in white clover (Bond and Webb 1989) and poplar (Lee-Stadelmann et al. 1989). "Thin cell layers" (Tran Than Van 1973) are well known to represent explants leading to unique morphogenetic responses (e.g. tobacco: Tran Than Van and Thrin 1986, rapeseed: Klimaszewska and Keller 1985, sugar beet:
43
Fig.6. Microtome cross section through the apical region of the
Amman M, Wanner G, Zenk MH (1986) Planta 167:310-320
seedling axis of a 12 day old seedling of barley (cv. "Igri").
Bond JE, Webb KJ (1989) Plant Sci. 61:119-125
Detrez et al. 1988, sunflower: Pelissier et al. 1990). Although the handsections used in these investigations do not represent typical "thin cell layers", it cannot be excluded that under conditions which are unknown as yet, qualitatively different responses from those of standard explants might be experienced. Microexplants allow identification of responding tissues at higher degrees of resolution. These investigations are an initial attempt at exploiting this approach for seedlings of barley. Sections from the apical part of the seedling axis (Fig. 5) showed the highest callus induction frequencies. Callus formation occured from all the tissues outside the central vascular elements, excluding however, the coleoptile and the scutellum. In contrast, the scutellum and coleoptile are known to be the responding tissues in immature embryo explants in a variety of Poaceae (Vasil and Vasil 1982, Lu and Vasil 1982, Lu et al. 1983, Ozias-Akins and Vasil 1982, 1983). Our future studies will concentrate on the plant regeneration capabilities of seedling axis-derived sections. We eventually hope to establish systems which are compentent for both transformation and regeneration.
Detrez C, Tetu T, Sangwan RS, Sangwan-Noreel BS (1988) J. Exp.
Acknowledgements The help of Dr. G. Wanner with the histological analysis is gratefully appreciated. The work was funded by BMFT (Bundesministerium fiir Forschung und Technologie), grant 0319398A.
Bot. 39:917-926 Dunwell JM (1986) Barley, In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of plant cell culture, vol IV, Macmillan Publishing Company, New York, pp 339-369 J~ihne A, Lazzefi PA, L6rz H (1991) Plant Cell Rep. 10:1-6 Jelaska S, Rengel Z, Cesar V (1984) Plant Cell Rep. 3:125-129 Klimaszewska K, Keller WS (1985) Plant Cell Tissue Organ Culture 4: 183-197 Lee-Stadelman OY, Loo SW, Hackett WP, Read PE (1989) Plant Sci. 61 : 263-272 Lu CY, Vasil IK (1982) Amer. J. Bot. 69:77-81 Lu CY, Vasil V, Vasil IlK (1983) Theor. Appl. Genet. 66:265-289 Mohanty BD, Ghosh PD (1988) Ann. Bot. 61:551-555 Ozias-Akins P, Vasil IK (1982) Protoplasma 110:95-105 Ozias-Akins P, Vasil IK (1983) Protoplasma 117:40-44 P61essier B, Bouchefra O, P@in R, Freyssinet G (1990) Plant Cell Rep. 9:47-50 Tran Than Van M (1973) Planta 115:87-92 Tran Than Van M, Thrln TH (1986)
In: Evans DA, Sharp WR,
Ammirato PV, Yamada Y (eds) Handbook of plant cell culture, vol IV, Macmillan Publishing Company, New York, pp 316-335 Vasil V, Vasil IK (1982) Bot. Gaz. 143:454-465 Wernicke W, Brettell R (1980) Nature 287:138-139 Wernicke W, Brettell R (1982) Protoplasma 111 : 19-27 Wernicke W, Milkovits L (1984) J. Plant Physiol. 115:49-58 Wernicke W, Brettell R, Wakizuka T, Potrykus I (1981) Zeitschr.
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