effect of culture medium and light conditions on the morphological characteristics and carbohydrate...

ACTA PHYSIOLOGIAE

PLANTARUM Vol. 20. No. 4. 1998:383-392

Effect of culture medium and light conditions on the morphological

characteristics and carbohydrate contents of Medicago strasseri calli

Marta Medina*, Nieves Villalobos, Pedro J. De La Cruz, ** Ana Dorado***, Hilario Guerra*

Departamento de Biologia Vegetal (Fisiologfa Vegetal), Facultad de Biologia, Universidad de Salamanca, Spain *Departamento de Biologia Vegetal (Fisiologfa Vegetal), Facultad de Farmacia, Universidad de Salamanca, Spain **Departamento de Biologia Vegetal (Biologfa General), Facultad de Biologia, Universidad de Salamanca, Spain ***Departamento de Estadistica y Matemfiticas Aplicadas, Facultad de Economia y Empresa,

Universidad de Salamanca, Spain

Key words: Medicago strasseri; carbohydrates; morphology; weight; t issue culture; callus

Abstract

Using 6 culture media (12, 12D, 12G, 11, A and B) made up of MS medium (Murashige-Skoog, 1962) supplemented or not with glycerine, with different cytokinins, and/or 2,4-D, the morphological characteristics and contents in total carbohydrates, reducing sugars, sucrose and starch were studied in calli induced from explants (cotyledon, petiole, hypocotyl and leaf) obtained from Medicago strasseri seedlings. Callus formation was induced under photoperiod (16h light/8h darkness) conditions or in the absence of light.

Considerable variability in the calli was observed, depending on the explants and media used. Under photoperiod conditions, medium A with KIN (1 mg/1) and 2,4-D (3 rag/l) induced many calli with the highest contents in total carbohydrates (886.1- 889.3 mg/g DW), sucrose (132.1-188.2 mg/g DW) and starch (125.2-247.6 mg/g DW) and the lowest contents in reducing sugars (118.4-173.3 mg/g DW). In media 11, A and B, under conditions of darkness, calli degenerated at the start of culture. Calli developed in darkness generally had dry weights and total carbohydrate and starch contents lower than those cultured under photoperiod conditions. However, sucrose contents were greater in calli formed in darkness.

At these cultures times, differentiation, in the form of organogenesis, was only seen using medium B with cotyledons, petioles and leaves as explants. It was also observed when petioles

were cultured in medium A but with a less pronounced organogenic response.

List of abbreviations: C.H., carbohydrates ; D, darkness; 2,4-D, 2 ,4-dichlorophenoxyacet ic acid; DW, dry weight; FW, fresh weight; KIN, kinetin; Ph, photoperiodic conditions; TDZ, Thidiazuron.

Introduction

Medicago strasseri Greuter, Matth~is and Risse, was described in 1982 (Greuter et al. 1982) as a new species by a German t eam f rom the Botanical Gar- dens in Berlin. The seeds were originally collected in Crete by the same G e r m a n team that described the species. In 1983, the Depar tment of Plant Biol- ogy of the Higher Technical School of Agricultural Engineer ing in Madrid received a small sample of seeds of this species f rom the Berlin Botanical Gar- dens. As f rom that year, and f rom the same seed sample, the species was mult ipl ied until a broad collection of Medicago strasseri plants became

available.

Medicago strasseri is a legume of great interest f rom the point of view of forage owing to its resistance to

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3,4. MEDINA, N. VILLALOBOS, P.J. DE LA CRUZ, A, DORADO & 14. GUERRA

winter cold and because it offers an alternative for animal nutrition in zones with continental climate. It has been proposed that large scale introduction of the species could serve to reduce feed imports.

The difficulty involved in the use of seeds to propa- gate the species together with the fact that commercial micropropagation cannot be applied to this type of cultivation owing to the high costs of the ma- nipulation mean that the production of artificial seeds could offer considerable practical and eco- nomic advantages.

Wi th the final aim of collecting somatic embryos of Medicago strasseri, in designing the present work we thought it essential to begin our research with callus formation since this is a crucial part of the development of organised structures such as organs or embryos.

Many factors affect in vitro callus formation: type of explant used;environmental conditions of the culture and, above all, the phytohormones added to the medium.We tested 4 types of explants - cotyledons, petioles, hypocotyls and leaves - and 6 culture media based on MS (Murashige-Skoog 1962) medium supplemented or not with glycerine, with different cytokinins and/or with 2,4-D, as specified in Materials and Methods. With these as tools, our aim was to determine which type of explant and which type of cytokinin are able to induce calli in Medi- cago strasseri and to elucidate the characteristics of each type of callus produced.

Since carbohydrates serve as sources of carbon and energy in callus formation (Rawal et al. 1984, Swarnkar et al. 1986) and since the characteristics of calli are largely determined by their carbohydrate composition (Keller et al. 1988, Naidu and Kishor 1995, Vu et al. 1995), we studied these com- pounds in the different calli formed to determine which medium/a and composition(s) are the most suitable for callus induction.

Materials and Methods

Plant material

Explants were established from cotyledons, petioles, hypocotyls and leaves of seedling of M.

strasseri (Greuter et al. 1982). Seeds were provided by the Department of Plant Physiology of the Higher Technical School of Agricultural Engineer- ing in Madrid, Spain.

Seeds were germinated on solid MS medium without sucrose and with salt concentration at one quar- ter strength. Seeds were kept in a dark chamber at 23 +_2 °C. When hypocotyl reached a height of 10 cm, the plantlets were transferred to a chamber with a 16 h photoperiod (20 pE-m-l.s q ) and planted for 20 - 30 days.

The different explants (1 cm slices of cotyledons, petioles, hypocotyls and leaves) were incubated in chambers with a 16 h photoperiod (20 ~tE.m-l.s q) or in complete darkness for four months at 23 +2 °C on solid MS medium (agar medium) supplemented or not with glycerine, with different cytokinins and/or with 2,4-D, as specified in Table 1. After four months of culture, calli were obtained. These were sufficiently large to provide the necessary material for studies on total carbohydrates, reducing sugars, sucrose and starch.

Table 1. Compositions of culture media used.

Added com- Culture medium pounds (mg/l) to MS medium 12 12D 12G 1 1A B

2,4-D 2.00

TDZ 0.01 0.001 0.01 1.00

KIN

Glycerine 30 000

3.00 2.00

1.00 2.00

Measurements dry and fresh weights

To determine dry weight, callus cultures on the different media were weighed and then placed in an oven at 40 °C until constant weight was attained; this was considered dry weight.

Extraction and evaluation of total carbohydrates,

reducing sugars and sucrose

The method used for extraction was based on that of Nguyen and Paquin (1971), with some modifications.

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EFFECT OF CULTURE MEDIUM AND LIGHT ,..

0.5 g of calli from each culture medium was rapidly washed in an aqueous solution of 60 mM polyeth- yleneglycol and then washed several times with distilled water. Then calli were ground down in a mortar with 5 ml of 95 % ethanol and filtered in vacuo with Albet n ° 1305 filter paper. The filtrates were recovered. Residues were washed again with 70 % ethanol, filtered and both filtrates were mixed. 3 ml of distilled water was added to the mixture plus 4 ml of chloroform. The mixture was then shaken and stored for 14 h at 4 °C, thereafter collecting the upper ethanolic phase.

To evaluate total carbohydrates, the phenol- sulfuric acid method was used (Dubois et al. 1956). Briefly, 0.5 ml of distitledwater and 0.5 ml of 5 % phenol were added to 100 gl of dry ethanolic extract. After shaking, 2.5 ml of concentrated H2SO 4 was added. The mixture was left to stand for 30 min and absorbance was read at 490 nm. The results were determined according to standard line obtained with different concentrations of D-glucose subjected to the same procedure.

Reducing sugars were evaluated using the method of Somogyi (1952) and Nelson (1944). 200 gl of distilled water plus 200 ~1 of Somogyi reagent were added to 100 gl of ethanolic extract, once dried. The mixture was boiled for 20 min and, after cooling, 200 ~1 of Nelson reagent plus 2.4 ml of distilled water were added. The mixture was shaken to elimi- nate CO 2 and absorbance was measured at 540 nm. The results were determined according to standard line obtained with different concentrations of D- glucose subjected to the same procedure.

Sucrose levels were evaluated following the method described by Paek et al. (1988). 100 ~1 of a solution of invertase (10 mg of commercial invertase in 10 ml of 0.2 M sodium acetate buffer, pH 4.5) plus 100 gl of distilled water were added to 100 pl of ethanolic extract and this was heated in a water bath at 55 °C for 10 min. After this time, 200 ~tl of Somogyi reagent was added and the mixture was boiled for 20 min. Then, 200 ~1 of Nelson reagent was added plus 2.4 ml of distilled water. Finally, the mixture was shaken and absorbance was measured at 540 nm. Sucrose concentrations were determined by calculating the difference between the measurement of sugars obtained when the enzyme was

added to the extract and the measurement of reducing sugars initially present in the extract.

E x t r a c t i o n a n d eva lua t ion o f s tarch

This was performed following the method of Gor- don et al. (1986) with slight modifications.

0.5 g of calli was boiled in 10 ml of 80 % ethanol for 2 or 3 min. After boiling, the preparation was filtered in vacuo using Albet n ° 1305 paper. The resi- due was dried in an oven at 30 °C until constant weight was attained and then ground in a mortar to a fine powder. This powder was used for starch evaluation.

The powder obtained from each sample was di- vided into two equal parts which were placed in centrifuge tubes. 4 mt of distilled water was added to each tube and the tubes were incubated for 1 h at 100 °C. After they had cooled, 1 ml of a solution of amyloglucosidase (25 mg of commercial amyloglucosidase was dissolved in 1 ml of 0.05 M sodium acetate buffer) was added to one of the tubes. This was spun down at 3000 g for 5 rain and the super- natant was passed through 10 ml Bio-Cel gel col- umns, before being brought up to 25 ml with the same buffer. In the case of the other tube (used as control), 1 ml of 0.05 M sodium acetate buffer, pH 4.5was added. Both tubes (covered with aluminium foil) were incubated for 8 h at 50 °C. After this time, they were allowed to cool and were then centri- fuged for 10 min at 3000 g, after which the super- natants were recovered

For starch evaluation, 0.5 ml of each sample was incubated for lh at 30 °C with 2 ml of an enzymic solution prepared with 8 ~tl of glucose-6-phospho- dehydrogenase (G-6-PDH) and 10 ~al of hexokinase in 50 ml of enzymic buffer. The enzymic buffer consisted of a mixture of 3.4 g of imidazole, 4.06 g of MgC12, 800 ml of distilled water and 0.2 g of bo- vine serum albumin. This mixture was adjusted to pH 6.9 with HC1 and brought up to 1 1 with distilled water, then adding 750 mg of NAD and 600 mg of ATR This mixture was kept at 4 °C until use).

After the incubation time, absorbance was determined at 340 nm. The results were determined according to standard line obtained with different

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M. MEDINA, N. VILLALOBOS, P.J. DE LA CRUZ, A. DORADO & H. GUERRA

concentrations of starch, subjected to amyloglucosidase treatment and evaluated as described above.

T h e following were used as blanks: a) 2 ml of enzymic buffer and 0.5 ml of distilled water; b) 2 ml of enzymic solution and 0.5 ml of extract incubated without amyloglucosidase; c) 2 ml of enzymic solution, 0.25 ml of distilled water and 0.25 ml of the amyloglucosidase solution; d) 2 ml of distilled water and 0.5 ml of extract incubated with amyloglucosidase, and e) 2 ml of enzymic solution and 0.5 ml of a solution of potato starch (Sigma) (0.1 g in 100 ml of distilled water).

Statistical assays

Results were analysed statistically using the analy- sis of variance included the SPSS program. When ANOVA showed treatment effects (p.01 and p.05), the least significant difference (Fisher LSD) test was applied to compare means at the 0.01 and 0.05 levels of significance.

Results and Discussion

Culture under photoper iod condit ions (16h light,

-8h darkness)

A s shown in Table 2, most of the calli obtained under photoperiod conditions were friables and greenish. Calli formed from hypocotyls and leaves in media 12, 12D and A and those from cotyledons in media 12, 12D and B were light green with dark green points, which seemed to indicate that in this calli organogenesis or embryogenesis could have taken place. However, only a clear and important organ formation has taken place (12 _+1.5 to 23 ___3.1 per callus) from cotyledons, petioles and leaves culti- vated in medium B. Taking the petiole in medium A the former answer of differentiation was also gener- ated, but the number of organs per callus was very small (3 +0.6). These calli were characterized as well for having a greenish colour with dark green points and were friable (except for those obtained from petioles in medium B, which were compact.

Cal lus fresh weight (Table 3) varied considerably, depending on the type of explant and culture medium used. Very small calli were formed from coty-

ledons and leaves in medium 12 G and from leaves in medium B. The largest calli were obtained using hypocotyls as explants cultured in medium 12.

Table 2. Morphological characteristics of calli formed in different media under photoperiodic conditions.

Culture Explant media

Morphological characteristics

Texture Colour

12 Cotyledon Friable greenish, d. green points

Petiole Compact greenish, witish

Hypocotyl Friable greenish, d. green points

Leaf Compact greenish, d. green points

12D Cotyledon Friable greenish, d. green points

Petiole Compact d. green, brownish zones

Hypocotyl Friable greenish, d. green points

Leaf Friable greenish, d. green points

12G Cotyledon Compact

Petiole Friable

Hypocotyl Friable

Leaf Compact

d. green, brownish zones

greenish, brownish zones

greenish, d. green points

greenish, witish

11 Cotyledon Friable

Petiole Friable

Hypocotyl Friable

Leaf Friable

greenish

greenish

greenish,

greenish

witish

A Cotyledon Friable

Petiole Friable

Hypocotyl Friable

Leaf Friable

greenish,

greenish,

greenish,

greenish,

witish

d. green points

d. green points

d. green points

Cotyledon Friable

Petiole Compact

Hypocotyl Friable

Leaf Friable

greenish,

greenish,

greenish,

greenish,

d. green points

d. green points

witish

d. green points

d. = dark

The amount of dry matter in the calli formed in the different media was very small (Table 4). In most calli, it ranged between 30 and 40 mg of DW. The lowest DW value was found in calli obtained in medium 11 using cotyledons as explants and in those obtained from petioles in medium 11 and from hypocotyls in medium 12D. The highest DW contents were observed in calli induced from hypocotyls and

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EFFECT OF CULTURE MEDIUM AND LIGHT...

Table 3. Fresh weight (g) of calli formed in different media. D, darkness; Ph, photoperiodic conditions. Each result shows the average of three replicates: experiments were repeated three times.

Explants Culture media

12 12D 12G 11 A B

Ph D Ph D Ph D Ph Ph Ph

Cotyledon 9.31_+0.9 3.98_+0.3 11.64_+0.4 8.04_+0.5 2.40_+0.1 3.56_+0.2 13.43+0.8 9.05±1.1 11.09_+04

Petiole 9.05_+1.0 7.59_+0.7 12.12_0.5 11.37_+0.5 9.41_+1.0 3.57±0.1 3.86±0.4 8.07±0.6 10.65_+0.6

Hypocotyl 14.54_+0.8 5.73_+0.4 7.08_+0.2 2.93_+0.4 9.64_+0.3 3.86_+0.3 5.52_+0.3 4.65_.+0.3 9.62_+0.6

Leaf 5.75_+0.3 7.31_+0.7 13.11_+0.7 8.68_+0.6 2.56_+0.1 5.89_+0.2 13.64_+0.1 7.09+_0.5 2.64_+04

leaves in medium 12G and in those formed in medium B using hypocotyls as explants. Overall, with all the types of explant used callus DW was greater when medium 12 G was used. The addition of glycerol to the culture medium thus seems to induce greater synthesis of material perhaps, as suggested by Vu e ta l . (1993), owing to a large accumulation of starch. However, as detailed below, in calli formed in this medium starch is not the main compound accumulated.

As may be seen from Fig. 1, most of the dry weight of the calli was in the form of total carbohydrates. As regards the different explants and media used, no strong differences in the carbohydrate levels of the calli were observed: values ranged between 812,4 and 903 mg/g DW. Fig. 1 also shows that medium A seems to induce high and very similar levels of total carbohydrates, regardless of the explant used.

Vu et al. (1995) emphasized that the presence of glycerol in the culture media induces high carbohydrate levels in calli. However, the addition of glycerol to our culture media (12 G medium) afforded a similar or lower carbohydrate content, in calli from leaves, than that observed for the rest of the media used.

Reducing sugars are rapidly accumulated during callus formation (Swarnkar e t al. 1986) since the sucrose present in the medium is hydrolysed by wall-bound invertases and it is glucose and fructose that are taken up by the cell (Komor et al. 1981). According to our own observations (Fig. 1), callus reducing sugar levels vary depending on the type of explant and medium used. The highest levels were seen in calli induced from hypocotyls cultured in

medium 12 D and the lowest in calli derived from cotyledons and hypocotyls cultured in medium A. Fig. 1 also shows that calli formed in medium A contain the lowest levels of reducing sugars. As shown in Fig. 2, reducing sugars represent between 13.4 (cotyledons and hypocotyls cultured in medium B) and 31% (bypocotyls cultured in medium 12 D) of the total carbohydrates in the different calli.

In calli, sucrose can be stored as a temporary reserve and its hydrolysis into hexoses may provide the necessary substrate for growth (Venkataramana e t al. 1991) and the synthesis of reserve materials such as starch (Huber and Azakawa 1991). Fig. 1 shows callus sucrose contents. Strong variations in the sucrose levels detected can be seen, depending on the type of explant and medium used. Extreme values were obtained in calli from leaf and hypocotyl explants cultured in medium 12 and in those derived from cotyledons and petioles incubated in medium A. With a few exceptions, medium A seems to induce high callus sucrose contents. Me- dium 12 favours a low accumulation of sucrose in calli.

Regarding total carbohydrate contents (Fig. 2), sucrose contents range between 1.2 % in calli from leaves cultured in medium 12 and 18.4 % in calli from petioles cultured in medium B.

Starch is usually accumulated in calli, regardless of the culture conditions employed. However, the amount of starch stored varies considerably, depending on the type of callus formed (Branca e t al.

1994). This is confirmed by the results observed in our work (Fig. 1). Starch was detected in all the calli but its contents varied strongly, depending on the

387


10N ~l)h TOTAL C.H. ] lO00 ~-D TOTAL C.H. ] • Coty~do, Fig. 2. Percentage of reducing sugars, sucrose and :INI starch wi thin total carbohydra tes contents . D, dark- i ~ ~ I~ ~ I X y i l ~ ° t s J l ness; Ph, photoperiodic conditions. ,n,, a,, sod

~ "-- [ ] I Le . f

0 ~ 0 - - "

2D|:~C;UILTURE11 AMEDIAB 12 CULTUREI2D MEDIA'~" ,~ 40 / e h REDILff::ING SUGAR~ ~' 50 DFD~'~'DUCING SLliGARS ]

E ~ 100 100 ~ 0 0

12 IZDI~G !1 A H 12: 12D 12[; 0 " ' " 0 " " " CULTURE HEDIA CULTURE HEDIA

12 IZDlZG I I A O 12 12D 12[; ~ SO I~,h ~l lrnn,~r ~ 30 . . . . . . . . . CULTURE MEDIA MEDIA CULTURE

. ~ [ P h SUCROSE J ~ n SUCROSE '~

-,00 | ,°

[ 0 ~' 0 12 12012G 11 A n lZ 120 IZG 1Z 1 2 D I ~ !1 A B IZ 12D 126 CULTUREMEDIA CULTUREMEDIA

CULTURE MEDIA CULTURE MEDIA

IPh STARCH S00 D STARCH

12 12DiSh I I A B 12 12D 19n CULTURE MEDIA CULTURE MEDIA

4o rh STARCH ~ "~ ./ I i 20

12 12012[; I I A B 12 12D 12[; CULTURE MEDIA CULTURE MEDIA

Fig. 1. Total carbohydrates, reducing sugars, sucrose and starch contents of calli formed in different media. D, darkness; Ph, photoperiodic conditions. Each point shows the average of three replicates: experiments were repeated three times. Vertical bars indicate SE.

type of explant and medium used. The greatest amount of starch was evaluated in calli derived from leaves cultured in medium A but there was also a strong accumulation of starch in calli obtained from hypocotyls cultured in medium 12 and from cotyledons cultured in media 12 and A. This strong accumulation of starch is not surprising since, for example, the starch content in nucellar calli from Citrus s inensis L. accounts for approxi- mately 30 % of the dry weight (Vu etal . 1993). This strong accumulation of starch in calli may be in- dicative of later callus development and differentiation. Working with tobacco, in 1995, Naidu and Kishor observed that organ-forming calli have higher starch contents than those that do not. There are also many works which have shown that starch accumulation in callus cells is a prerequisite for in

vitro shoot development (Thorpe et al. 1986, Stamp, 1987) and even for the development of ~5- matic embryogenesis, although it cannot be considered a determinant factor in embryogenesis (Keller et al. 1988).

Further scrutiny of Fig. 1 reveals the very low starch contents evaluated in calli from hypocotyls sown in medium B. Although starch accumulation seems to be a general phenomenon, the low amounts detected in some of our calli do not seem to be very abnormal since Lai and McKersie (1994) reported collecting petiole calli with starch contents below 20 mg/g dry weight. It is interesting that in calli in which organogenesis occurs - such as those obtained from cotyledons, petioles and leaves in medium B - starch levels are low, as reported by Mangat et al. (i990) for organogenesis. These

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E F F E C T O F C U L T U R E M E D I U M A N D L I G H T ...

Table 4. Dry weight (mg/g fresh weight) of calli formed in different media. D, darkness; Ph, photoperiodic conditions. Each result shows the average of three replicates: experiments were repeated three times.

Explants Culture media

12 12D 12G 11 A B

Ph D Ph D Ph D Ph Ph Ph

Cotyledon 9.31-+0.9 3.98+_0.3 11.64+_0.4 8.04_+0.5 2.40+0.1 3.56-+0.2 13.43+_0.8 9.05+1.1 11.09-+04

Petiole 9.05+_1.0 7.59_+0.7 12.12+0.5 11.37_+0.5 9.41+1.0 3.57-+0.1 3.86_+0.4 8.07+0.6 10.65+-0.6

Hypocotyl 14.54+_0.8 5.73-+0.4 7.08+-0.2 2.93+0.4 9.64+0.3 3.86+0.3 5.52_+0.3 4.65+_0.3 9.62-+0.6

Leaf 5.75+_0.3 7.31+-0.7 13.11+_0.7 8.68+_0.6 2.56_+0.1 5.89_+0.2 13.64-+0.1 7.09+_0.5 2.64+_04

authors considered that since organogenesis re- quires considerable amounts of energy, this may de- rive from the degradation of starch. The low starch contents evaluated in organogenic calli in medium B are also seen in other calli that have still not un- dergone differentiation, although it is not possible to rule out the idea that such differentiation may oc- cur later on during development and that the calli may already have differentiation potential.

In view of the proportion of callus starch (Fig. 2) in total carbohydrate contents, again strong variation was seen since the values range between 29.7 % in- calli f rom hypocotyls cultured in medium 12 and 0.6 % in calli f rom hypocotyls cultured in medium B.

Culture in darkness

It should be stressed that under conditions of darkness it is only possible to elicit callus formation using media 12, 12D and 12G. With media 11, A and B, in the darkness, calli did begin to form but then rapidly degraded. In the darkness the above- described differentiation for photoperiod conditions did not take place.

T h e morphological characteristics of the calli are described in Table 5. Most of the calii were friables, except those obtained f rom cotyledons and leaves cultured in medium 12G, which were compact, and those obtained from hypocotyls in medium 12, which were soft. Calli were not greenish but rather whitish and yellowish. In two of the calli (fi'om cotyledons and leaves cultured in medium 12G) the colour was brownish, possibly due to callus degen- eration.

Cal lus fresh weight (Table 3) ranged between 2.93 g (obtained from hypocotyls sown in medium 12D) and 11.37 (obtained from petioles cultured in medium 12D) Considering the three culture media, hypocotyls seemed to be outstanding as explants, owing to the production of calli of relatively low weight, and also leaves, because they gave rise to calli of relatively high weight. However, in all calli dry weight was very low (Table 4). Although it would be hard to generalise, cotyledons seem to in-

Table 5. Morphological characteristics of calli formed in different media under darkness.

Culture Explant media

Morphological characteristics

Texture Colour

12 Cotyledon Friable yellowish

Petiole Friable yellowish

Hypocotyl Softness whitish

Leaf Friable yellowish

12D Cotyledon Friable yellowish


Hypocotyl Friable whitish

Leaf Friable yellowish

12G Cotyledon Compact brownish


Hypocotyl Friable yellowish

Leaf Compact brownish

duce calli with the highest D W values while petioles afford those with the lowest DW.

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Total carbohydrate levels were high (Fig. 1). The contents of reducing sugars (Fig. 2) range between 13.7 % (when petioles were used as explants and culture was carried out in medium 12) and 38.4 % (when culture was performed with hypocotyls in medium 12G) with respect to total carbohydrate contents. As may be seen in Fig. 1, hypocotyls sown in medium 12G afforded the explants that induced calli with the highest amounts of reducing sugars per g of DW, in agreement with the work of Vu et al.

(1995) for "Hamlin" orange tissue cultures using a medium with glycerol. However, this cannot be ex- trapolated to all the calli obtained in medium 12G using other explants.

A s seen in Figure 1, the highest sucrose contents were observed in calli formed from cotyledons and petioles cultured in medium 12, from hypocotyls cultured in medium 12D, and from leaves cultured in medium 12G. Regarding total carbohydrates (Figure 2), callus sucrose contents ranged between 7.2 % (from petioles cultured in medium 12D) and 24.1% (from hypocotyls cultured in medium 12D).

Starch synthesis in calli is a rapid process that occurs at the start of their formation (Swarnkar et al. 1986, Branca et al. 1994). Callus starch contents vary considerably. As shown in Figure 2, describing the proportions of starch in calli with respect to total carbohydrate contents, values as low as 0.7 % (calli from cotyledons cultured in medium 12) and as high as those found for calli derived from petioles cultured in medium 12D were observed. Figure 1 shows one explant -hypocotyl - that yielded mini- mum starch levels in the three culture media and underscores one medium -medium 12- which induces calli with contents lower than any other medium using any type of explant.

Comparison o f cultures made under photoperiod conditions and in the darkness

It is important to compare the results obtained under photoperiod conditions and under darkness so that future studies on callus development and differentiation can be based on data that will shed light on the requirement or not of a photoperiod prior to transferring the culture to photoperiod conditions, as reported by Zheng et al. (1966).

Tables 2 and 5 show a difference as regards colour between calli formed in the darkness and those derived under photoperiod conditions. Calli obtained under photoperiod conditions were greenish, some of them with dark green points, while those formed in darkness were more whitish or yellowish. In some cases, the texture of the calli also varied.

Overall (Table 3) the fresh weight of calli induced in darkness was lower than those formed under photoperiod conditions. In only three case the formation of calli with greater fresh weight was important: those induced from leaves cultured in medium 12 and those from cotyledons and leaves cultured in medium 12G. Working with lignous plants, Rout et

al. (1995) achieved better callus formation in darkness than under the photoperiod conditions used here. Our results partly conflict with those authors' observations (Rout et al. 1995) since, with the exception of the three above cases, photoperiod conditions induced greater callus formation. Unlike fresh weight, it is not possible to draw a general idea from the results obtained for dry weight (Table 4) because they vary depending on the explant and culture medium used.

Overall, total carbohydrate contents were lower in calli induced in darkness (Fig. 1). Only one explant - that derived from leaves - induced calli with a higher carbohydrate content in darkness in both medium 12D and 12G. Medium 12 G was also striking; with the exception of hypocotyls, this medium induced calli with higher amounts of carbohydrates.

As regards reducing sugars (Fig. 1 ), no clear differences can be seen between calli developed in the darkness and those obtained under photoperiod conditions. In calli obtained in darkness and in medium 12, with the exception of cotyledons, a lower amount of reducing sugars was seen. However, medium 12G, except for leaves, induced calli in darkness with similar or greater amounts of carbohydrates than those obtained under light conditions. One type of explant was singular in the results; whatever the medium used in darkness cotyledons produced calli with greater amounts of reducing sugars.

When calli were induced in darkness, their sucrose content generally increased (Fig. 1). Only three ex-

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E F F E C T O F C U L T U R E M E D I U M A N D L I G H T .,.

plants -petioles and leaves in medium 12D and hypocotyls in medium 12G - produced calli with lower sucrose contents in darkness than under photoperiod conditions.

Lower starch contents were found in calli developed in darkness than in those obtained under photoperiod conditions (Fig. 1). In view of the high sucrose levels obtained in calli formed in darkness, as in Jenner (1982) and Preiss (1992), sucrose can be considered to be the main source of starch synthesis and since sucrose is not used lower starch levels in calli are produced. From the results obtained on starch produced under conditions of darkness, three cases were outstanding (petioles in medium 12D and petioles and leaves in medium 12G) in which the starch contents of the calli were greater than those observed under photoperiod conditions. In these cases, with the exception of calli from petioles cultured in medium 12D, the sucrose content was higher in darkness than under photoperiod conditions. Contrary to the above suggestion, low sucrose concentrations accompanied by high levels of starch in dark-cultured calli, there seems to be no relationship between high callus sucrose contents and low starch contents. However, it should be stressed that petioles and leaves in medium 12G, which contains glycerol, produced the highest starch contents in calli developed in darkness. In these cases, the formation of starch in calli may oc- cur at the expense of glycerol, as pointed out by Vu et al. (1993).

C o n c l u s i o n s

The conclusions that can be drawn from the present work are as follows:

- The best medium for callus growth, considered as the dry weight content, is medium 12 G (MS plus TDZ and glycine).

- The best medium for callus differentiation is medium B (MS plus 2 mg/L of 2,4 D and 2 mg/L of KIN).

- The best explant for callus differentiation is the petiole

- The best hormones for the induction of callus differentiation are 2,4 D with KIN.

- The best illumination conditions for differentiation are photoperiod conditions (16 h light/8 h darkness).

- The calli produced in organogenesis, considering accumulated dry matter (30 mg/g FD), are small.

- The main characteristic of calli in which organogenesis will take place is their low starch content.

Statistical study

Comparison of the results obtained on measuring the amount of total carbohydrates, reducing sugar, sucrose and starch of the calli statistically, for the different explants, culture media and illumination conditions employed, revealed highly significant differences (p<0.01).

A c k n o w l e d g e m e n t s

This work was supported by a grant from the Direc- cidn General de Investigacidn Cientffica y T6cnica, Spain, N ° PB94-1403.

R e f e r e n c e s

Branca C., Torelli A., Fermi P., Altamura M.M., Bassi M. 1994. Early phases "in vitro" culture tomato cotyledony: Starch accumulation and protin partten in relation to the horizontal treatment. Protopiasma 182, 59-64.

Dubois M., Gilles K.J., Smith F. 1956. Colorimetric method for determination of sugars and related sub- stances. Anal. Chem. 28,356-361.

Gordon A.J., Ryle G.J.A., Mitchell D.F., Lowry K.H., PoweU C.E. 1986. The effect of defoliation on carbohydrate, protein and leg-haemoglobin content of white clo- ver nodules. Annals of Botany 58, 141-154.

Greuter W., Matthfis U., Risse H. 1982. Notes on Car- daegean plants. 3. Medicago strasseri, a new leguminous shrub from Kriti. Willdenowia 12,201-206.

Huber S.C., Akazawa T. 1986. A novel sucrose syn- thase pathway for sucrose degradation in cultured syca- more cells. Plant Physiol. 81, 1008-1013.

Jenner C.F. 1982. Storage of Starch. In: Loevus FA and Tanner W (eds) Plant carbohydrates I, Intracellular carbohydrate, pp. 700-737. Springer-Verlag. Berlin.

Keller G.L., Nikolau B.J., Ulrich T.H. Wurtele E.S. 1988. Comparison of starch and ADP-glucose pyro- phosphorylase levels in nonembryogenic ceils and de-

391


veloping embryos from induced carrot cultures. Plant Physiol. 86, 451-456.

Komor E., Thorn M., Maretzki A. 1981. The mecha- nism of sugar uptake by sugarcane suspension cells. Planta 153, 181-192.

Lai F-M., McKersie B.D. 1994. Regulation of starch and protein accumulation in alfalfa (Medicago sativa L.) somatic embryos. Plant Sci. I00, 211-219.

Mangat B.S., Pelekis M.K., Cassells A.C. 1990. Changes in the starch content during organogenesis in in vitro cultured Begonia rex stem explants. Physiol. Plant. 79, 267-274.

Murashige T., Skoog F. 1962. A revised medium for rapid growth and bioassays with tabacco tissue cultures. Physiol. Plant. 15,473-497.

Naidu K.R., Kishor P.B.K. 1995. Activities of hydro- lytic enzymes in callus cultures of tobacco during or- ganog6nesis. J. Bioscien. 20, 629-636.

Nelson N. 1944. A photometric adaptation of the So- mogyi method for the determination of glucose. J. Biol. Chem. 153,375-380.

Nguyen S.T., Paquin R. 1971. M6thodes d' extraction et de purification des acides amin6s libres et des protdines de tissus vdg6taux. J. Chromatogr. 61,349-351.

Paek K.Y., Canderlerd S., Thorpe T.A. 1988. Physio- logical effects of Na2SO4 and NaCI on callus cultures of Brassica campestris ( Chinesse cabbage). Physiol. Plant. 72, 160-166

Preiss J. 1982. In: Loevus FA and Tanner W (eds) Plant carbohydrates I, Intracellular carbohydrate, pp. 397- 417. Springer-Verlag. Berlin.

Rawal S.K., Dwivedi U.N., Khan B.M., Masearenhas A.F. 1984. Biochemical aspects of shoot differentiation

in sugarcane callus: Carbohydrate metabolizing enzymes. J. Plant Physiol. 119, 191-199.

Rout G.R., Samantaray S, Das P. 1995. Somatic embryogenesis and plant regeneration from callus culture of Acacia catechu - a multipurpose leguminous tree. Plant Cell, Tiss. Org. Cult. 42, 283-285.

Somogyi M. 1952. Notes on sugar determination. J. Biol. Chem. 195, 19-23.

Stamp J.A. 1987. Somatic embryogenesis in cassava. The anatomy and morphology of the regeneration process. Ann. Bot. 57,451-459.

Swarnkar P.L., Bohra S.P., Chandra N. 1986. Bio- chemical studies on initiation of callus in Solanum surat- tense. J. Plant Physiol. 126, 293-296.

Thorpe T.A., Joy R.W., Leung D.W.M. 1986. Starch turnover in shoot-forming tobacco callus. Physiol. Plant. 66. 58-62.

Venkataramana S., Naidu K.M., Singh S. 1991. Inv¢r- tases and growth factors dependent sucrose accumulation in sugarcane. Plant Sci. 74, 65-72.

Vu J.C.V., Niedz R.P., Yelonsky G. 1993. Glycerol stimulation of chlorophyll synthesis, embryogenesis, and carboxylation and sucrose metabolism enzymes in nucellar callus of "Hamlin" sweet orange. Plant Cell, Tiss. Org. Cult. 33, 75-80.

Vu J.C.V., Niedz R.P., Yelonsky G. 1995. Activities of sucrose metabolism enzymes in glycerol grown suspension cultures of sweet orange (Citrus sinensis L. Os- beck). Env. Exp. Bot. 35,455-462.

Zheng, Q., Dessai, A.P., Prakash, C.S. 1996. Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis. PI. Cell. Rep. 15,381-385.

Received October 28, 1997; accepted September 24, 1998

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