Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol160.Issue2.2001:

(1)

Hormonal status of maize initial explants and of the embryogenic

and non-embryogenic callus cultures derived from them as related

to morphogenesis in vitro

Vı´ctor M. Jime´nez

_{, Fritz Bangerth *}

Institut fur Obst-,Gemu¨se-und Weinbau(370),Uni6ersita¨t Hohenheim,D-70593Stuttgart,Germany

Received 24 February 2000; received in revised form 26 June 2000; accepted 5 September 2000

Abstract

Endogenous hormone levels (indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins1, 3and20[GAs], zeatin/zeatin riboside

[Z/ZR] andN6_[_D2_{-isopentenyl] adenine}_/_N6_[_D2_{-isopentenyl] adenosine; [iP}_/_{iPA]) were analysed in immature maize zygotic embryos}

of two maize (Zea mays L.) genotypes, known for their distinct ability to generate embryogenic (E) callus. No differences were found among genotypes in the hormone contents of the embryos. These embryos were also used as initial explants to establish callus cultures. E and non-embryogenic (NE) calli were obtained from the competent genotype (A188), while only NE callus was produced by the incompetent one (B73). The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. When analysing the endogenous hormone levels in the various callus types generated in each genotype, it was found that only differences in the IAA levels accounted for variations in the morphogenic properties of the calli. Higher levels of endogenous IAA were typical of embryogenic callus cultures. It was also observed, that a loss in the embryogenic competence of the calli, due to a prolonged time of culture, occurred concomitantly with a reduction in the IAA levels, practically to the levels found in the non-embryogenic calli. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Phytohormones; Radio-immunoassay; Somatic embryogenesis; In vitro culture;Zea mays(L.)

www.elsevier.com/locate/plantsci

1. Introduction

Green and Phillips [1] were the first to report the successful regeneration of complete plants from maize tissue cultures. In all previous attempts only non-competent callus was obtained. Since then, techniques and culture media adequate for stimu-lating the embryogenic response in this plant have been improved, as well as the comprehension of the mode of action of several factors that influence the establishment, growth and differentiation of embryogenic callus cultures (reviewed by Bajaj

[2]). Although regenerable maize callus cultures can be initiated from many meristematic tissues of the plant, immature embryos are the best source of callus with regeneration competence [2].

Different callus types can be induced in maize, i.e. Type I, II and NE callus [3]. Type-I callus is generally white and compact and seems to be a further advanced differentiation step of Type-II callus. This latter type is soft, white or pale yellow, friable, can usually retain totipotency after long periods of time in culture, and is similar to the embryogenic cultures of model plant species such

as Daucus and Nicotiana spp. [4 – 6]. NE callus is

translucent, does not show any sign of organisa-tion and produces only roots when the 2,4-D concentration in the culture medium is reduced [7]; this is the typical callus obtained in non-embryo-genic lines [6].

* Corresponding author. Tel.: +49-711-4592350; fax: + 49-711-4592351.

E-mail address:[email protected] (F. Bangerth).

1_{Present address: CIGRAS, Universidad de Costa Rica, 2060 San}

Pedro, Costa Rica.

(2)

As in tissue culture of most plant species, in maize, the genotype plays a very important role in the establishment, growth and subsequent differ-entiation of callus cultures. There are several re-ports in which differences among genotypes in callus formation rates have been found in this species [4,8 – 11].

Some attempts have been carried out in certain species to associate the endogenous hormone lev-els of explants and cultures derived from them with their regeneration competence [12 – 14]. To the best of our knowledge, the only report in maize is that of Carnes and Wright [15], in which they investigated endogenous hormones, such as indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin (Z), zeatin riboside (ZR),N6_[_D2_{-isopentenyl]}

adenine (iP) and N6_[_D2_{-isopentenyl] adenosine}

(iPA), in immature kernels of genotypes reported to have different levels of competence.

To understand the hormonal regulation during somatic embryogenesis in maize might permit the application of this developmental process to a broader spectrum of genotypes, which may be very useful for clonal propagation, multiplication of F1 hybrids and especially genetic transformation in maize [7]. Efficient procedures for obtaining sus-pension cultures and protoplasts from embryo-genic callus, which are capable of plant regeneration, enables cell technologies to be ap-plied, along with conventional methods, for speed-ing up the selection process and makspeed-ing it easier [16].

The aims in this study were to establish embryo-genic and non-embryoembryo-genic callus lines from com-petent and incomcom-petent maize genotypes and to measure and relate endogenous hormone contents in these lines to their embryogenic competence. Furthermore, the endogenous hormone levels of the immature zygotic embryos and the endosperm of competent and incompetent genotypes were compared.

2. Material and methods

2.1. Plant material

Seeds of the maize (Zea mays L.) inbred lines A188 (Minnesota Agricultural Experiment Sta-tion, 1948) and B73 (Iowa State University, 1972) were obtained from the Versuchsstation fu¨r

Pflanzenzu¨chtung Eckartsweier, Universita¨t Ho-henheim, Willsta¨tt, Germany. The seeds were sown in 0.5-l pots, in vermiculite, and grown in a growth chamber (12 h photoperiod [275 mmol m−2 _s−1_{] provided by Osram HQI-T 400W/D}

lamps [Munich, Germany] and 20°C constant tem-perature) for three to four weeks (until the plants reached a 4 – 5-leaves stage). At this time, the plantlets were transplanted to 51 pots in a 1:1 mixture of sand and commercial plant growth substrate (Klasmann-Deilmann GmbH, Geeste-Groß Hesepe, Germany) and grown under natural light in a greenhouse maintained at 22/18°C day/ night; additional light during the winter was pro-vided by the same lamps named above. When the plantlets were three weeks old, a weekly fertilisa-tion program with 2 ml l−1 _{of Wuxal (complete}

nutrient solution) (Aglukon GmbH, Du¨sseldorf, Germany), was started. Four to eight plants per line were established per experiment, and a total of four experiments were carried out during the years 1997, 1998 and 1999.

To avoid cross pollination, the ear shoots were covered (bagged) before the silks (styles) emerged. When the silks were visible (in 60 – 64 days old A188 plants and a week later for B73 plants), the tips of the husks and silks were cut off with a sharp knife (‘cutting back’) according to the proce-dure described by Neuffer [17] and pollinated daily during the following 3 – 4 days. Under the condi-tions used, the plants were ready to be harvested for the establishment of in vitro cultures after 85 – 95 days.

2.2. Culture media

The callus induction medium (KK+_{) contained}

the N6 salts and vitamins [18], 3% sucrose, 2.3 g l−1 _{proline, 0.1 g l}−1 _{enzymatic casein}

hy-drolysate, 2 mg l−1 _{2,4-D and 15.3 mg l}−1

AgNO3. pH was adjusted to 6.0 with KOH before

autoclaving. All components were added prior to autoclaving except for AgNO3, which was filter

sterilised [19]. The Petri dishes containing the in-duction culture medium were stored in the dark immediately after the media solidified, and briefly exposed to light only during the explanting pro-cess, to avoid photodegradation of AgNO3. For

callus maintenance the previous medium without AgNO3 was employed (KK−).

(3)

For embryo development, segments of the es-tablished callus cultures were transferred to MS (Murashige and Skoog [20]) medium, supple-mented with 0.5 mg l−1_{thiamine HCl, 0.5 mg l}−1

pyridoxine HCl, 0.5 mg l−1_{nicotinic acid, 2.0 mg}

l−1 _{glycine, 100 mg l}−1 _{myo-inositol and 3%}

sucrose — called R1 medium — and incubated under light (4.75 W m−2_{) provided by Philips}

(Eindhoven, Netherlands) TL 40 W/33 RS fluores-cent lamps. The pH of this medium was adjusted to 5.8 with KOH before autoclaving.

2.3. In 6itro establishment of immature embryos

The tips of the ears were removed 15 – 18 days after pollination. Then the ears were cut in half, and both sections were surface-sterilised with a 2.5% sodium hypoclorite solution containing two drops of Tween 20 (10 min), and finally washed three times with sterile deionised water. Immature embryos were excised from the seeds under sterile conditions (following the procedure of Armstrong [21]) — the tips of several rows of kernels were cut off with a scalpel, without touching the embryo, and the endosperm was then scooped out of the kernel using the back of the blade. The embryo was then removed from the detached endosperm and the length determined on a ruled scale, and placed on 25 ml of KK+ _{nutrient medium in}

90×15 mm Petri dishes (30 embryos per dish) with the embryo axis in contact with the solid medium. For establishment and maintenance, cul-tures were incubated at 26°C in the dark. Four experiments were carried out with more than 800 embryos dissected in this way.

2.4. Hormone content of immature embryos and

endosperm

To determine, if the endogenous hormone levels of embryos and endosperm, play a role in the morphogenetic characteristics of the calli devel-oped from the first, embryos and endosperms were excised, 15 DPA, as previously described, and analysed for hormone contents as will be outlined below.

2.5. Subcultures

Two weeks after culture initiation (CI) the new formed callus was separated from remnants of the

original embryo tissues and from germinated coleoptiles and roots, and transferred to KK−

medium. After two additional weeks of culture, the calli were classified into embryogenic (E) and non-embryogenic (NE) under a dissecting micro-scope and subcultured separately. Subsequent sub-cultures were done every 2 to 4 weeks; and on each subculture the explants were classified again as E and NE callus, and large callus segments were divided into fragments of approximately 3 mm. Cultures were incubated under the above men-tioned maintenance conditions.

To characterise differences in the in vitro be-haviour between the lines A188 and B73, the responses of all the explants were evaluated weekly during the first 7 weeks of culture.

2.6. Hormone content of embryogenic and

non-embryogenic calli

After 7 and 13 weeks of culture under the above mentioned maintenance conditions, samplings were carried out to evaluate differences in the endogenous hormone content of the embryogenic and non-embryogenic calli. Simultaneously, part of the E and NE calli were transferred to regener-ation conditions to evaluate their regenerregener-ation ca-pacity. The second evaluation (after 13 weeks) was carried out to evaluate an eventual loss in the regeneration capacity and to try to relate it to changes in the hormone levels.

2.7. Sampling, extraction and purification of plant

hormones

The determination of IAA, ABA, gibberellins1, 3

and 20(GAs), Z/ZR and iP/iPA was performed on

the same sample. Samples of the different materi-als were surface dried and cleaned with a paper towel, immediately frozen in liquid nitrogen and stored at −20°C. The frozen samples were then freeze-dried, and stored again at −20°C until extracted.

Samples (approximately 300 mg dry weight [DW]) were ground in liquid nitrogen, ho-mogenised and then extracted overnight with 50 ml 80% cold aqueous methanol (B4°C) in dark-ness at 4°C. The extracts were then filtered through G4-glassinter-filters (max. pore size 10 – 16

mm; Schott, Mainz, Germany). To control the losses that can occur during the purification

(4)

proce-dure, an internal standard of 1-14_{C-IAA (spec. act.}

14 mCi mmol−1_{; Amersham, Braunschweig,}

Ger-many) was added at this point. The extract was dried under low pressure and dissolved in 12 ml 0.1 M ammonium acetate (pH 9.0) by using an ultrasonic bath and subsequently frozen at −20°C overnight. After thawing, the extract was centrifuged at 22 000 rpm and 4°C for 25 min.

For purification, the supernatant from the cen-trifugation step was passed through a precondi-tioned column combination of poly-vinylpyrrolidone (Sigma Chemical Co., Deisen-hofen, Germany), DEAE-Sephadex A-25 (Phar-macia, Freiburg, Germany) and a C18 Sep-Pak

cartridge (reversed phase, Waters, Eschborn, Germany) (modified after Bertling and Bangerth [22]).

2.8. Quantification of hormones

Hormones were quantified by radio-im-munoassay with polyclonal antibodies. Fractions containing IAA, ABA and GAs were methylated with diazomethane. Antibodies used were raised in rabbits against free IAA, ABA, GA3, ZR and iPA

and radioimmunological hormone analysis was performed according to Bohner and Bangerth [23]. Cross reactions of the GA3 antibody used were

determined by Bertling and Bangerth [22], to be about 90% with GA1 and GA20. Therefore, the

GAs determined by means of this antibody are expressed as GA3 equivalents and called GAs in

the following text.

Overall recovery during the described purifica-tion procedures was previously determined with radiolabelled internal standards, and was found to be between 40 and 70% for IAA, 92% or higher for ABA, higher than 89% for GAs and than 80% for both cytokinins. Therefore, the IAA internal standard was the only one used regularly and the IAA levels obtained were adjusted with the corre-sponding recovery value for each sample, due to the high variations found.

2.9. Statistical analysis

Endogenous hormonal levels were determined in at least three biological replications and analysed using the STATISTICA for Windows (StatSoft Inc., Tulsa, OK, USA) Version 5.1 Student-Ver-sion; ’97 Edition. The Post-Hoc Tukey’s

Honest-Significant-Difference-Test (HSD) for unequal N (Spjotvoll/Stoline) was used to determine signifi-cant differences in hormone levels means (B0.05).

3. Results

3.1. Comparati6e analysis of callus response

Of the dissected A188 embryos, the length of 366 of them was determined and gave an average of 2.5990.58 mm; on the other hand, the 723 embryos of the line B73 measured 2.3490.50 mm. Close examination, using the dissecting micro-scope, did not show any difference in the develop-mental stage of the sampled embryos of both lines. Since embryos were placed with the embryo axis in contact with the culture medium, development of scutellum callus was stimulated [3].

The evaluation of the morphogenetic responses of the immature embryos of A188 and B73 to the culture conditions, during the first 7 weeks after CI, is presented in Fig. 1. Immature embryos of both lines, responded to the culture on medium by increasing in size during the first 2 days. After that, they started germinating precociously at a very high rate (over 80%), but simultaneously began forming callus. The growth rate of these incipient calli was impaired by the more dominant growth of the coleoptile, which often occurs in a rotated or corkscrew manner. After the germi-nated structures were discarded (2 weeks after CI), leaving only remains of the scutellum and callus segments, the growth rate of the latter increased. At the second subculture (4 weeks after CI), the calli were classified, according to their morpho-genetic characteristics, as non-organised translu-cent (NE) and compact with discernible structures (E), and cultured on separate Petri dishes.

The characteristics of the calli produced, varied among genotypes. In line A188, embryogenic com-pact callus (similar to the type I, described by Armstrong and Green [3]) formed at a high rate, with few NE callus development. Meanwhile, the B73 line originated practically only NE callus. NE callus was characterised by being completely fri-able and translucent, without any sign of organisa-tion, or of root development.

The type-I callus obtained included some sec-tions with a high profusion of meristematic centres that sometimes germinated. Trying to maintain

(5)

this callus in an undifferentiated state, the germi-nating structures were eliminated on each subcul-ture. During the last 4 weeks evaluated, and concomitantly with a reduction in the percentage of calli with embryogenic characteristics, an in-crease in the amount of NE callus in line A188 was recognised, reaching almost 30% at the end of the evaluation period (Fig. 1).

It is also important to note that some highly organised and friable small callus segments ap-peared in the A188 cultures after 3 weeks in culture. These callus segments were very similar to the type-II callus described by Armstrong and Green [3]). Although they were isolated and sub-cultured separately, it was not possible to raise the amount of tissue necessary to carry out hormone analyses, as a way to compare this callus type with the type-I callus.

Due to the reduction in the amount of type-I callus during the time mentioned above (Fig. 1), it was decided to sample part of the cultures for hormone analysis immediately after the seventh evaluation. At the same time, some callus

seg-ments were transferred to the regeneration condi-tions mentioned above and evaluated after 2 weeks, to measure their morphogenetic capacity. This was repeated 6 weeks later (13 weeks after CI), to evaluate an eventual loss in the competence of the E callus, as described elsewhere [24].

None of the NE samples, neither from A188 nor from B73, showed any sign of regeneration com-petence on any of the evaluation dates, just some root growth in approximately one third of the samples. In contrast, 100% of the calli classified as E, and sampled after seven weeks of culture, showed morphogenetic capacity, whereas none of the calli sampled after 13 weeks of culture regener-ated at all, which is evidence for a loss in the regeneration capacity of type-I callus as a result of a prolonged time in culture.

In view of these results, the maize calli were then classified into three categories according to their morphogenetic characteristics: those com-petent calli still maintaining their regeneration ca-pacity at the sampling date (E+), those E calli that had already lost their competence at the time

Fig. 1. Morphogenetic in vitro responses of immature embryos of maize lines A188 and B73 during the first 7 weeks of culture. A subculture was carried out after every even evaluation.

(6)

Fig. 2. Endogenous levels of IAA (A), ABA (B), GAs (C), Z/ZR (D) and iP/iPA (E) in embryos and endosperm of maize lines A188 and B73, 15 – 18 days after pollination. Significant differences (B0.05) between lines are marked with distinct letters.

of sampling (E−), and calli that did not show any morphogenetic capacity from the beginning (NE). The endogenous hormone contents of calli in each of these categories were analysed and associated with their morphogenetic properties.

3.2. Hormone content of immature embryos and

endosperm

The endogenous hormone levels in immature zygotic embryos and endosperm of maize lines A188 and B73, 15 DPA, are presented in Fig. 2. No statistically significant differences were found in the levels of IAA between A188 and B73 imma-ture embryos. In contrast, higher levels of this hormone were measured in the endosperms of line B73 than in those of line A188. The endogenous levels of all other hormones evaluated were similar in both varieties in embryo- and endosperm-tissue.

3.3. Endogenous hormone le6els in the callus

cultures

The hormone contents of the different maize

calli types, analysed by radioimmunoassay, are shown in Fig. 3. The E+ calli of the line A188 contained higher levels of IAA than the other callus types, all of them non-competent at the sampling date. No differences, concerning this hormone, were found between the calli that never reached embryogenic competence (NE) and those that lost it over the time of culture (E−). ABA levels were highest in E calli that had lost their embryogenic capacity (E−), followed by the other two callus types of the same line A188 (E+ and NE), and the lowest levels were found in the NE callus of the non-competent line B73. Higher GAs levels were found in the E+ calli, lower in the NE ones, and intermediate levels (non-different from any of the two extremes) in the E− calli. The lowest Z/ZR levels were found in the still com-petent callus (E+), with the highest in the E−, and intermediate levels in those calli that never showed embryogenic response (NE of both lines). iP/iPA levels in E+ callus were higher than those in the NE calli of line B73; levels not significantly different from them were found in the other two callus types of line A188 (E- and NE).

(7)

4. Discussion

4.1. Induction, maintenance and regeneration of in

6itro cultures

Lines A188 and B73 were chosen to conduct this work, because they were reported to have a very different behaviour when their immature em-bryos are cultured in vitro [4,25,26]. Line A188 (agronomically poor) has proved to be the best source for the establishment of embryogenic cul-tures of maize [1,4,11,21,27]. On the other hand, line B73 has shown a poor in vitro response, when compared with other genotypes [4,25]; although other reports indicate that, under certain circum-stances, embryogenic callus could be obtained from B73 immature embryos as well [10,19,28,29]. Under our experimental conditions, line A188 confirmed its distinction as a good E-callus-pro-ducing genotype, while 100% of the B73 explants produced NE callus. These results supported the use of these lines in the present study, for compari-son between a competent and a non-competent line.

However, even if the selection of the lines was suitable, this seemed not to be the case regarding the stage of development of the immature em-bryos. On one side, the precocious germination rate of the immature embryos used in this experi-ment was extremely high, and on the other, it was not possible to obtain a stable type-II callus line, despite successful reports on genotype A188 using the same culture conditions [30].

The stage of development is better characterised in maize by the size than by the age of the embryos, because maize embryos can reach the proper size anywhere from 9 to 15 DPA [31]. It has been reported that embryos 1 – 2 mm in size produced maximal values of callus development, while embryos bigger than 2 mm often germinated without callus formation [16,21,32]. Large em-bryos (2 – 3 mm) were used intentionally in this work, because one of the aims was to compare the endogenous hormone levels in the explants from which callus originates (in the same stage of devel-opment) with the morphogenetic characteristics of the derived callus. Since almost 1000 embryos at

Fig. 3. Endogenous levels of IAA (A), ABA (B), GAs (C), Z/ZR (D) and iP/iPA (E) in E+ and NE (7 weeks after CI) and in E- (13 weeks after CI) maize callus cultures, grown in the presence of 2,4-D. Significant differences (B0.05) are marked with distinct letters.

(8)

this stage were necessary to conduct the hormone analyses, the use of smaller embryos would have increased the amount of embryos to be isolated, which would have been very laborious.

After discarding the germinated structures of the embryos, callus formation and growth were more evident. In this work, NE callus was ob-tained in both lines, but type-I callus just in line A188. When culturing non-embryogenic lines with 2,4-D, usually just NE callus, a soft, watery, yel-lowish and non-morphogenetic callus, is formed [6,16]; on the other hand, competent lines are typified by the growth of both E and NE callus [33]. The type-I calli are usually compact, white, non-friable, regenerate through both somatic em-bryogenesis and organogenesis and are highly dif-ferentiated (often contain leaf-like structures and advanced-stage somatic embryos on maintenance medium) [16,21]. In this work, we observed that the amount of type-I callus was reduced along the time of culture (Fig. 1). Mo´rocz et al. [34] reported a similar event in the same callus type, that ulti-mately reduced the amount of callus available. Dolgykh [16] mentioned that it is difficult to keep type-I callus for prolonged time in culture, because the growth of the callus slows down as a conse-quence of the germination of the meristematic bodies. It is possible that under the culture condi-tions used by us (probably the 2,4-D concentra-tion), could not maintain all the somatic embryos in a competent but still repressed state, therefore they started to germinate.

After transferring to the regeneration conditions (MS medium without 2,4-D and providing the cultures with light), as expected, none of the NE callus-types regenerated. E callus sampled seven weeks after CI produced regenerants, while E calli transferred to regeneration conditions 13 weeks after CI did not regenerate. It has been observed that type-I callus quickly lose its competence. Vasil et al. [24] stated that the type-I callus can not usually be maintained for more than three subcul-tures (9 – 10 weeks) because a rapid and extensive differentiation of somatic embryos occurs or be-cause it turns brown and grows very slowly, there-fore losing its regeneration capacity.

4.2. Hormone content of immature embryos and

endosperm

Genotypic differences in morphogenesis may be

due to differences in endogenous hormone levels and to the uptake and metabolism of the plant growth regulators added to the culture medium [35]. However, in the present study, no differences were found in the endogenous hormone levels of embryos (the initial explants) of maize genotypes with different morphogenetic capacity. Apart from higher levels of endogenous IAA in the endosperm of the non-competent line B73, no other differ-ences were found in the levels of the other hor-mones evaluated. Contrarily, higher levels of IAA were found by the authors analysing the en-dosperm of a competent wheat genotype than in a non-competent one [36]. However, endosperm seems not to play a very important role in confer-ring embryogenic competence to the immature zygotic embryos. In addition, contrary to what we observed in wheat, a species in which higher en-dogenous ABA levels in the initial explants were found in the embryogenic genotype than in the non-embryogenic one, in maize, such a difference was not apparent. This variation between species, correlated to the behaviour observed during the initial steps of culture of the immature embryos under in vitro conditions. While very high and similar rates of precocious germination were ob-served soon after culture initiation in both maize genotypes (Fig. 1), very low rates were found in the wheat genotype with the higher ABA levels [36].

As previously mentioned, to the best of our knowledge, there is only one previous work, in which endogenous hormone levels in maize kernels of different genotypes were related to their compe-tence to develop embryogenic callus [15]. There a 16 – 20-fold less total kernel auxin concentration in A188 than in the less competent line Missouri-17 was found. They hypothesised that very high en-dogenous auxin levels may produce a similar in-hibitory effect in competent callus formation, as culturing the maize embryos in very high 2,4-D levels. However, the levels of auxins in the whole kernels may poorly reflect the levels in the imma-ture embryos, as observed in wheat [36] and barley [37]. Since the endosperm constitutes the majority of kernel dry matter, its endogenous hormone levels (when expressed on a DW basis) would mask those of the embryos that are, in fact, the initial explants. As can be observed in Fig. 2, the high endogenous IAA levels in the endosperm of

(9)

the less competent genotype are not present in the corresponding embryos. Lur and Setter [38], analysing maize endosperm, found low levels of IAA accumulation at 9 DPA that abruptly in-creased until reaching a peak at 20 DPA.

Contrasting with the results of the present study, in which no differences were found in the level of endogenous cytokinins between genotypes, Carnes and Wright [15] observed that the whole A188 kernels contained around 50% less Z/ZR than the Missouri-17 kernels. They correlated the lack of embryogenic competence in the latter genotype to superoptimal cytokinin levels. Lur and Setter [38] found a maximal concentration of Z/ZR in maize endosperms at 9 DPA, coinciding with maximal rates of cell division, and then de-clined abruptly between 9 and 15 DPA. Also, Wenck et al. [39], reported that low endogenous levels of cytokinins were characteristic of leaves of Dactylis glomerata with high embryogenic capac-ity. Centeno et al. [40] found similar contents of total cytokinins in cotyledons of three Corylus avellana genotypes with different levels of embryo-genic capacity; however, they showed a very differ-ent iP-type/Z-type cytokinin ratio. They reported that higher endogenous levels of iP and lower of Z correlated with higher levels of E competence, when compared with those of less competent genotypes.

4.3. Endogenous hormone le6els in the callus

cultures

To the best of our knowledge, there are no previous reports in maize, in which the relation of endogenous hormone levels in callus cultures to their morphogenetic capacity has been related.

The differences in the endogenous IAA levels found between the different callus types resemble those found in wheat [36]. The E callus that still maintained the competence to regenerate, con-tained higher levels of IAA than the other callus types. These results seem to confirm the previous conclusions in wheat, that high levels of IAA may play a crucial role in maintaining the regeneration capacity of callus cultures, at least in monocotyle-donous species.

The higher levels of ABA found in line A188, in comparison to those of line B73, could be geno-type-dependent because no pattern, in relation to the embryogenic competence, was observed.

Kop-ertekh and Butenko [13], analysed the hormone levels in wheat calli of embryogenic and non-em-bryogenic genotypes, without selecting among the callus types present in each genotype. They also found higher ABA levels in callus of the more competent variety than in that of the less com-petent one. It was also observed that the embryo-genic calli, that lost their competence over the time of culture, contained higher levels of ABA. This increase in the endogenous ABA levels may be one reason for the loss of competence, besides the strong decrease in the IAA contents. An inhibition of growth of maize cells of the cv. Black Mexican Sweet, was observed after the addition of ABA to the culture medium by Balsevich et al. [41].

Significantly higher levels of endogenous GAs were found in embryogenic callus, when compared with non-embryogenic. However, in the few re-ports dealing with the role of endogenous GAs levels in somatic embryogenesis, negative ([42] in carrot and [43] in geranium), as well as neutral roles ([44] in orchardgrass), have been postulated for this hormone group.

The absolute hormone levels may, by them-selves, not explain all the morphogenetic differ-ences between genotypes and callus cultures. The sensitivity of the genotypes to the hormones may have also played an important role in the differ-ences observed in the development between both lines. Dolgykh [16] has already postulated that differences between genotypes may be caused by differential response of the cells to exogenous plant growth regulators, mainly to 2,4-D. Bron-sema et al. [45] reported that one of the major differences between the more and the less com-petent lines in maize is the distribution of 2,4-D within the embryos, after culturing them on medium containing 2,4-D. They also informed that 2,4-D-uptake was more reduced in NE imma-ture embryos than in the E embryos on culimma-ture medium, and that most of the 2,4-D absorbed in both was not metabolised. They also found that the pattern of 2,4-D distribution within the em-bryos was different in cultured emem-bryos of both cultivars, and that cells located in regions with high accumulation of 2,4-D did not proliferate. In the present study, higher endogenous IAA levels could reflect the ability of maize callus cul-tures to express embryogenic competence. It was also observed that a reduction in this embryogenic competence correlated with a diminution in the

(10)

IAA levels. Endogenous hormone levels in the initial explants could not be associated to embryo-genic competence in the two genotypes evaluated.

Acknowledgements

V.M.J. was recipient of a German Academic Exchange Service (DAAD) Postgraduate Scholarship.

References

[1] C.E. Green, R.L. Phillips, Plant regeneration from tissue cultures of maize, Crop Sci. 15 (1975) 417 – 420. [2] Y.P.S. Bajaj, Biotechnology in maize improvement, in:

Y.P.S. Bajaj (Ed.), Maize, Biotechnology in Agriculture and Forestry, vol. 25, Springer, Berlin, 1994, pp. 3 – 23. [3] C.L. Armstrong, C.E. Green, Establishment and mainte-nance of friable, embryogenic maize callus and the in-volvement of L-proline, Planta 164 (1985) 207 – 214.

[4] T.K. Hodges, K.K. Kamo, C.W. Imbrie, M.R. Beckwar, Genotype specificity of somatic embryogenesis and re-generation in maize, Bio/Technology 4 (1986) 219 – 223. [5] P.F. Fransz, J.H.N. Schel, Ultrastructural studies on callus development and somatic embryogenesis in Zea maysL, in: Y.P.S. Bajaj (Ed.), Maize, Biotechnology in Agriculture and Forestry, vol. 25, Springer, Berlin, 1994, pp. 50 – 65.

[6] F.B.F. Bronsema, W.J.F. van Oostveen, A.A.M. van Lammeren, Comparative analysis of callus formation and regeneration on cultured immature maize embryos of the inbred lines A188 and A632, Plant Cell Tissue Organ Cult. 50 (1997) 57 – 65.

[7] A.M.C. Emons, H. Kieft, Somatic embryogenesis in maize (Zea mays L.), in: Y.P.S. Bajaj (Ed.), Somatic Embryogenesis and Synthetic Seed II, Biotechnology in Agriculture and Forestry, vol. 31, Springer, Berlin, 1995, pp. 24 – 39.

[8] M. Nesˇticky´, F.J. Nova´k, A. Piovarci, M. Doleoelova´, Genetic analysis of callus growth of maize (Zea maysL.) in vitro, Z. Pflanzenzu¨cht. 91 (1983) 322 – 328.

[9] D.R. Duncan, M.E. Williams, B.E. Zehr, J.M. Wid-holm, The production of callus capable of plant regener-ation from immature embryos of numerous Zea mays

genotypes, Planta 165 (1985) 322 – 332.

[10] D.T. Tomes, O.S. Smith, The effect of parental genotype on initiation of embryogenic callus from elite maize (Zea mays L.) germplasm, Theor. Appl. Genet. 70 (1985) 505 – 509.

[11] R. Tuberosa, C. Lucchese, Long-term totipotent callus cultures in the maize inbred B79, Agric. Med. 119 (1989) 412 – 416.

[12] K. Sasaki, K. Shimomura, H. Kamada, H. Harada, IAA metabolism in embryogenic and non-embryogenic carrot cells, Plant Cell Physiol. 35 (1994) 1159 – 1164.

[13] L.G. Kopertekh, R.G. Butenko, Naturally occurring phytohormones in wheat explants as related to wheat morphogenesis in vitro, Russ. J. Plant Physiol. 42 (1995) 488 – 491.

[14] J.R. Hess, J.G. Carman, Embryogenic competence of immature wheat embryos: genotype, donor plant, envi-ronment, and endogenous hormone levels, Crop Sci. 38 (1998) 249 – 253.

[15] M.G. Carnes, M.S. Wright, Endogenous hormone levels of immature corn kernels of A188, Missouri-17, and Dekalb XL-12, Plant Sci. 57 (1988) 195 – 203.

[16] Y.I. Dolgykh, Establishment of callus cultures and re-generation of maize plants, in: Y.P.S. Bajaj (Ed.), Maize, Biotechnology in Agriculture and Forestry, vol. 25, Springer, Berlin, 1994, pp. 24 – 36.

[17] M.G. Neuffer, Growing maize for genetic studies, in: M. Freeling, V. Walbot (Eds.), The Maize Handbook, Springer, New York, 1993, pp. 197 – 209.

[18] C.C. Chu, C.C. Wang, C.S. Sun, Establishment of an efficient medium for another culture of rice through comparative experiments on the nitrogen sources, Sci. Sinica 18 (1975) 659 – 668.

[19] D.D. Songstad, C.L. Armstrong, W.L. Petersen, AgNO3

increases type II callus production from immature em-bryos of maize inbred B73 and its derivatives, Plant Cell Rep. 9 (1991) 699 – 702.

[20] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture, Phys-iol. Plant 15 (1962) 473 – 497.

[21] C.L. Armstrong, Regeneration of plants from somatic cell cultures: applications for in vitro genetic manipula-tion, in: M. Freeling, V. Walbot (Eds.), The Maize Handbook, Springer, New York, 1993, pp. 663 – 671. [22] I. Bertling, F. Bangerth, Changes in hormonal pattern of

the new growth of Sclerocarya birreaafter rejuvenation treatment with GA3and ‘heading back’,

Gartenbauwis-senschaft 60 (1995) 119 – 124.

[23] J. Bohner, F. Bangerth, Effects of fruit set sequence and defoliation on cell number, cell size, and hormone levels of tomato fruits (Lycopersicum esculentumMill.) within a truss, Plant Growth Regul. 7 (1988) 141 – 155. [24] V. Vasil, I.K. Vasil, C. Lu, Somatic embryogenesis in

long-term callus cultures of Zea mays L. (Gramineae), Am. J. Bot. 71 (1984) 158 – 161.

[25] C.L. Armstrong, J. Romero-Severson, T.K. Hodges, Improved tissue culture response of an elite maize inbred through backcross breeding, and identification of chro-mosomal regions important for regeneration by RFLP analysis, Theor. Appl. Genet. 84 (1992) 755 – 762. [26] C. Rosati, P. Landi, R. Tuberosa, Recurrent selection

for regeneration capacity from immature embryo-derived calli in maize, Crop Sci. 34 (1994) 343 – 347. [27] R. Tuberosa, P. Landi, Analysis of callus proliferation

from immature embryos of maize genotypes, Agric. Med. 121 (1991) 51 – 55.

[28] K. Lowe, D.B. Taylor, P. Ryan, K.E. Paterson, Plant regeneration via organogenesis and embryogenesis in maize inbred line B73, Plant Sci. 41 (1985) 125 – 132. [29] A.S. Wang, D.S.K. Cheng, J.B. Milcic, T.C. Yang,

Effect of X-ray irradiation on maize inbred line B73 tissue cultures and regenerated plants, Crop Sci. 28 (1988) 358 – 362.

(11)

[30] P. Vain, H. Yean, P. Flament, Enhancement of produc-tion and regeneraproduc-tion of embryogenic type II callus in

Zea maysL. by AgNO3, Plant Cell Tissue Organ Cult. 18

(1989) 143 – 151.

[31] J.W. McCain, T.K. Hodges, Anatomy of somatic em-bryos from maize embryo cultures, Bot. Gaz. 147 (1986) 453 – 460.

[32] J.C. Sellmer, S.W. Ritchie, I.S. Kim, T.K. Hodges, Initiation, maintenance, and plant regeneration of type II callus and suspension cells, in: M. Freeling, V. Walbot (Eds.), The Maize Handbook, Springer, New York, 1993, pp. 671 – 677.

[33] B. Swedlund, R.D. Locy, Sorbitol as the primary carbon source for the growth of embryogenic callus of maize, Plant Physiol. 103 (1993) 1339 – 1346.

[34] S. Mo´rocz., G. Donn, J. Ne´meth, D. Dudits, An im-proved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspen-sion culture, Theor. Appl. Genet. 80 (1990) 721 – 726. [35] S. Bhaskaran, R.H. Smith, Regeneration in cereal tissue

culture: a review, Crop Sci. 30 (1990) 1328 – 1337. [36] V.M. Jime´nez, F. Bangerth, Endogenous hormone levels

in initial explants and in embryogenic and non-embryo-genic callus cultures of competent and non-competent wheat genotypes, in preparation.

[37] V.M. Jime´nez, F. Bangerth, In vitro culture and endoge-nous hormone levels in immature zygotic embryos, en-dosperm and callus cultures of normal and high-lysine barley genotypes, J. Appl. Biol. 74 (2000) in press. [38] H.-S. Lur, T.L. Setter, Role of auxin in maize endosperm

development, Plant Physiol. 103 (1993) 273 – 280.

[39] A.R. Wenck, B.V. Conger, R.N. Trigiano, C.E. Sams, Inhibition of somatic embryogenesis in orchardgrass by endogenous cytokinins, Plant Physiol. 88 (1988) 990 – 992.

[40] M.L. Centeno, R. Rodrı´guez, B. Berros, A. Rodrı´guez, Endogenous hormonal content and somatic embryogenic capacity of Corylus a6ellana L. Cotyledons, Plant Cell

Rep. 17 (1997) 139 – 144.

[41] J.J. Balsevich, A.J. Cutler, N. Lamb, L.J. Friesen, E.U. Kurz, M.R. Perras, S.R. Abrams, Response of cultured maize cells to (+)-abscisic acid, (−)-abscisic acid, and their metabolites, Plant Physiol. 106 (1994) 135 – 142. [42] M. Noma, J. Huber, D. Ernst, R.P. Pharis, Quantitation

of gibberellins and the metabolism of [3_{H]gibberellin A} 1,

during somatic embryogenesis in carrot and anise cell cultures, Planta 155 (1982) 369 – 376.

[43] M.J. Hutchinson, S. KrishnaRaj, P.K. Saxena, Inhibitory effect of GA3on the development of thidiazuron-induced

somatic embryogenesis in geranium (Pelargonium×hor

-torum Bailey) hypocotyl cultures, Plant Cell Rep. 16 (1997) 435 – 438.

[44] K. Rajasekaran, M.B. Hein, G.C. Davis, M.G. Carnes, I.K. Vasil, Endogenous growth regulators in leaves and tissue cultures ofPennisetum purpureumSchum, J. Plant Physiol. 130 (1987) 12 – 25.

[45] F.B.F. Bronsema, W.J.F. van Oostveen, E. Prinsen, A.A.M. van Lammeren, Distribution of [14_C]

dichlorophenoxyacetic acid in cultured zygotic embryos of Zea mays L, J. Plant Growth Regul. 17 (1998) 81 – 88.

(1)

3.2. Hormone content of immature embryos and endosperm

cultures

The hormone contents of the different maize

(2)

4. Discussion

4.1. Induction, maintenance and regeneration of in

6itro cultures

(3)

this stage were necessary to conduct the hormone analyses, the use of smaller embryos would have increased the amount of embryos to be isolated, which would have been very laborious.

4.2. Hormone content of immature embryos and endosperm

Genotypic differences in morphogenesis may be

(4)

4.3. Endogenous hormone le6els in the callus

cultures

To the best of our knowledge, there are no previous reports in maize, in which the relation of endogenous hormone levels in callus cultures to their morphogenetic capacity has been related.

The higher levels of ABA found in line A188, in comparison to those of line B73, could be geno-type-dependent because no pattern, in relation to the embryogenic competence, was observed.

(5)

IAA levels. Endogenous hormone levels in the initial explants could not be associated to embryo-genic competence in the two genotypes evaluated.

Acknowledgements

V.M.J. was recipient of a German Academic Exchange Service (DAAD) Postgraduate Scholarship.

References

[1] C.E. Green, R.L. Phillips, Plant regeneration from tissue cultures of maize, Crop Sci. 15 (1975) 417 – 420. [2] Y.P.S. Bajaj, Biotechnology in maize improvement, in:

[7] A.M.C. Emons, H. Kieft, Somatic embryogenesis in maize (Zea mays L.), in: Y.P.S. Bajaj (Ed.), Somatic Embryogenesis and Synthetic Seed II, Biotechnology in Agriculture and Forestry, vol. 31, Springer, Berlin, 1995, pp. 24 – 39.

[8] M. Nesˇticky´, F.J. Nova´k, A. Piovarci, M. Doleoelova´, Genetic analysis of callus growth of maize (Zea maysL.) in vitro, Z. Pflanzenzu¨cht. 91 (1983) 322 – 328.

[9] D.R. Duncan, M.E. Williams, B.E. Zehr, J.M. Wid-holm, The production of callus capable of plant regener-ation from immature embryos of numerous Zea mays genotypes, Planta 165 (1985) 322 – 332.

[10] D.T. Tomes, O.S. Smith, The effect of parental genotype on initiation of embryogenic callus from elite maize (Zea mays L.) germplasm, Theor. Appl. Genet. 70 (1985) 505 – 509.

[11] R. Tuberosa, C. Lucchese, Long-term totipotent callus cultures in the maize inbred B79, Agric. Med. 119 (1989) 412 – 416.

[12] K. Sasaki, K. Shimomura, H. Kamada, H. Harada, IAA metabolism in embryogenic and non-embryogenic carrot cells, Plant Cell Physiol. 35 (1994) 1159 – 1164.

[13] L.G. Kopertekh, R.G. Butenko, Naturally occurring phytohormones in wheat explants as related to wheat morphogenesis in vitro, Russ. J. Plant Physiol. 42 (1995) 488 – 491.

[14] J.R. Hess, J.G. Carman, Embryogenic competence of immature wheat embryos: genotype, donor plant, envi-ronment, and endogenous hormone levels, Crop Sci. 38 (1998) 249 – 253.

[15] M.G. Carnes, M.S. Wright, Endogenous hormone levels of immature corn kernels of A188, Missouri-17, and Dekalb XL-12, Plant Sci. 57 (1988) 195 – 203.

[17] M.G. Neuffer, Growing maize for genetic studies, in: M. Freeling, V. Walbot (Eds.), The Maize Handbook, Springer, New York, 1993, pp. 197 – 209.

[18] C.C. Chu, C.C. Wang, C.S. Sun, Establishment of an efficient medium for another culture of rice through comparative experiments on the nitrogen sources, Sci. Sinica 18 (1975) 659 – 668.

[19] D.D. Songstad, C.L. Armstrong, W.L. Petersen, AgNO3

increases type II callus production from immature em-bryos of maize inbred B73 and its derivatives, Plant Cell Rep. 9 (1991) 699 – 702.

[20] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture, Phys-iol. Plant 15 (1962) 473 – 497.

the new growth of Sclerocarya birreaafter rejuvenation treatment with GA3and ‘heading back’,

Gartenbauwis-senschaft 60 (1995) 119 – 124.

[23] J. Bohner, F. Bangerth, Effects of fruit set sequence and defoliation on cell number, cell size, and hormone levels of tomato fruits (Lycopersicum esculentumMill.) within a truss, Plant Growth Regul. 7 (1988) 141 – 155. [24] V. Vasil, I.K. Vasil, C. Lu, Somatic embryogenesis in

long-term callus cultures of Zea mays L. (Gramineae), Am. J. Bot. 71 (1984) 158 – 161.

for regeneration capacity from immature embryo-derived calli in maize, Crop Sci. 34 (1994) 343 – 347. [27] R. Tuberosa, P. Landi, Analysis of callus proliferation

from immature embryos of maize genotypes, Agric. Med. 121 (1991) 51 – 55.

Effect of X-ray irradiation on maize inbred line B73 tissue cultures and regenerated plants, Crop Sci. 28 (1988) 358 – 362.

(6)

[30] P. Vain, H. Yean, P. Flament, Enhancement of produc-tion and regeneraproduc-tion of embryogenic type II callus in Zea maysL. by AgNO3, Plant Cell Tissue Organ Cult. 18

(1989) 143 – 151.

[31] J.W. McCain, T.K. Hodges, Anatomy of somatic em-bryos from maize embryo cultures, Bot. Gaz. 147 (1986) 453 – 460.

[33] B. Swedlund, R.D. Locy, Sorbitol as the primary carbon source for the growth of embryogenic callus of maize, Plant Physiol. 103 (1993) 1339 – 1346.

culture: a review, Crop Sci. 30 (1990) 1328 – 1337. [36] V.M. Jime´nez, F. Bangerth, Endogenous hormone levels

in initial explants and in embryogenic and non-embryo-genic callus cultures of competent and non-competent wheat genotypes, in preparation.

development, Plant Physiol. 103 (1993) 273 – 280.

[39] A.R. Wenck, B.V. Conger, R.N. Trigiano, C.E. Sams, Inhibition of somatic embryogenesis in orchardgrass by endogenous cytokinins, Plant Physiol. 88 (1988) 990 – 992.

[40] M.L. Centeno, R. Rodrı´guez, B. Berros, A. Rodrı´guez, Endogenous hormonal content and somatic embryogenic capacity of Corylus a6ellana L. Cotyledons, Plant Cell Rep. 17 (1997) 139 – 144.

of gibberellins and the metabolism of [3_{H]gibberellin A} 1,

during somatic embryogenesis in carrot and anise cell cultures, Planta 155 (1982) 369 – 376.

[43] M.J. Hutchinson, S. KrishnaRaj, P.K. Saxena, Inhibitory effect of GA3on the development of thidiazuron-induced

somatic embryogenesis in geranium (Pelargonium× hor-torum Bailey) hypocotyl cultures, Plant Cell Rep. 16 (1997) 435 – 438.

[44] K. Rajasekaran, M.B. Hein, G.C. Davis, M.G. Carnes, I.K. Vasil, Endogenous growth regulators in leaves and tissue cultures ofPennisetum purpureumSchum, J. Plant Physiol. 130 (1987) 12 – 25.

[45] F.B.F. Bronsema, W.J.F. van Oostveen, E. Prinsen, A.A.M. van Lammeren, Distribution of [14_C]

dichlorophenoxyacetic acid in cultured zygotic embryos of Zea mays L, J. Plant Growth Regul. 17 (1998) 81 – 88.

Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol160.Issue2.2001:

Hormonal status of maize initial explants and of the embryogenic

and non-embryogenic callus cultures derived from them as related

to morphogenesis in vitro

Vı´ctor M. Jime´nez

_{, Fritz Bangerth *}

Parts

Dokumen yang terkait

PERENCANAAN PORTAL KELUAR KAMPUS III UMM

PERENCANAAN ULANG (REDESIGN) STRUKTUR ATAS PROYEK RUMAH SUSUN SEDERHANA 2 (RUSUNAWA) UMM MALANG DENGAN MENGGUNAKAN BALOK PRATEGANG PARSIAL

PERSEPSI MAHASISWA TENTANG MATERI TAYANGAN SANG PEMBURU DI LATIVI ( Studi Pada Mahasiswa Jurusan Ilmu Komunikasi UMM Angkatan 2002)

APRESIASI MAHASISWA TERHADAP TAYANGAN Â“OPERA VAN JAVAÂ” DI TRANS7 (Studi Pada Mahasiswa Jurusan Ilmu Komunikasi UMM Angkatan 2008)

FAKTOR-FAKTOR PENYEBAB KESULITAN BELAJAR BAHASA ARAB PADA MAHASISWA MA’HAD ABDURRAHMAN BIN AUF UMM

38 Mahasiswa UMM Pilih KKN di Padang

FPP UMM Seleksi Sarjana Membangun Desa

Lagi, UMM Tambah Mahasiswa Asing

Pengajian Muhammadiyah di UMM Akan Hadirkan BJ Habibie

Robert John Pope: UMM Miliki Intellectual Honesty

Dukungan

Links

Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol160.Issue2.2001:

Hormonal status of maize initial explants and of the embryogenic

and non-embryogenic callus cultures derived from them as related

to morphogenesis in vitro

Vı´ctor M. Jime´nez

, Fritz Bangerth *

Parts

Dokumen yang terkait

PERENCANAAN PORTAL KELUAR KAMPUS III UMM

PERENCANAAN ULANG (REDESIGN) STRUKTUR ATAS PROYEK RUMAH SUSUN SEDERHANA 2 (RUSUNAWA) UMM MALANG DENGAN MENGGUNAKAN BALOK PRATEGANG PARSIAL

PERSEPSI MAHASISWA TENTANG MATERI TAYANGAN SANG PEMBURU DI LATIVI ( Studi Pada Mahasiswa Jurusan Ilmu Komunikasi UMM Angkatan 2002)

APRESIASI MAHASISWA TERHADAP TAYANGAN Â“OPERA VAN JAVAÂ” DI TRANS7 (Studi Pada Mahasiswa Jurusan Ilmu Komunikasi UMM Angkatan 2008)

FAKTOR-FAKTOR PENYEBAB KESULITAN BELAJAR BAHASA ARAB PADA MAHASISWA MA’HAD ABDURRAHMAN BIN AUF UMM

38 Mahasiswa UMM Pilih KKN di Padang

FPP UMM Seleksi Sarjana Membangun Desa

Lagi, UMM Tambah Mahasiswa Asing

Pengajian Muhammadiyah di UMM Akan Hadirkan BJ Habibie

Robert John Pope: UMM Miliki Intellectual Honesty

Dokumen yang Anda mencari sudah siap untuk unduhkan

_{, Fritz Bangerth *}