Plant Cell, ~ssue and Organ Culture 44: 7-17, 1996. 7 (~) 1996 Kluwer Academic Publishers. Printed in the Netherlands.

High frequency somatic embryogenesis from coffee leaves Factors influencing embryogenesis, and subsequent proliferation and regeneration in liquid medium

Jos van B o x t e l 1 & M a r c B e r t h o u l y CIRAD-CP,, B.P. 5035, 34032 Montpellier, France (1present address: Dept. of Virus Research, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK)

Received 29 May 1995; accepted in revised form 14 August 1995

Key words: Coffea, embryogenic callus suspensions, leaf explants, regeneration

A b s t r a c t

An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called "high frequency" embryogenic callus of Coffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures. Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus 1-1 in a modified MS medium containing 4.5 #M 2,4-dichlorophenoxyacetic acid, under 3 #mol m -2 s- 1 illumination, and subcultured every 7-10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250-1000 #m in diameter) at an inoculum density of 5 g 1-1, with medium renewed every 3-4 weeks. Under these conditions, embryogenic potential of C. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro- and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g 1-1. Under these conditions of culture, 1 g of C. canephora or Arabusta callus produced 1.2 and 0.9 × 105 somatic embryos, respectively, after 8-10 weeks in liquid regeneration medium. This was an overall reduction of 4--6 months from explant to regenerant, when compared with other procedures.

Abbreviations: BA - N6-benzyladenine, HFSE - high frequency somatic embryogenesis, IAA - indole-3-acetic acid, IBA - indole-3-butyric acid, rpm - rotations per minute, LFSE - low frequency somatic embryogenesis, MS - Murashige & Skoog medium, PPF - photosynthetic photon flux, 2,4-D - 2,4-dichlorophenoxyacetic acid, 2-iP - 2-isopentenyladenine

I n t r o d u c t i o n

Various alternative procedures for tissue regeneration in coffee have been described, since somatic embryo- genesis from Coffea canephora P. ex Fr. internodes was first reported by Staritsky (1970). High frequency somatic embryogenesis (HFSE) was first reported by S/3ndahl & Sharp (1977) on leaves of Coffea arabi- ca L. 'Bourbon'. According to these authors, HFSE

is characterized by the abundant appearance of fri- able, highly embryogenic callus. They obtained such HFSE-callus after a two-step procedure: a primary cul- ture on auxin-containing medium for callus induction, followed by a second culture on auxin-free medium for embryo induction. The specific nature of HFSE-callus permits its use in liquid culture systems, as previously described by S/3ndahl et al. (1985) and Zamarripa et al. (1991). Because it rapidly generates large amounts of

Table 1. Composition of media used in experiments in mg 1- I.

C 1 E l CP R EG 2 DEV 3

macrominerals MS4/2 MS/2 MS/2 MS/2 MS/2 MS

microminerals MS/2 MS/2 MS/2 MS/2 MS/2 MS

FeSO4.7H20 13.9 13.9 13.9 13.9 13.9 27.8

Na2EDTA 18.65 18.65 18.65 18.65 18.65 37.3

thiamine-HCl 10 20 5 10 8 1

pyridoxine-HCl 1 - 0.5 1 3.2 1

nicotinic acid 1 - 0.5 1 - 1

calcium pantothenate . . . . . 1

biotin . . . . . 0.01

glycine 1 20 - 2 - -

L-cysteine - 40 10 - - -

myo-inositol 100 200 50 200 100 100

adenine sulfate - 60 - 40 - -

casein hydrolysate 100 200 100 400 - -

malt extract 400 800 200 400 - -

2,4-D 0.5 1 1-2 - - -

IBA 1 . . . . .

IAA . . . . 0.45 -

2-iP 2 . . . . .

kinetin - - 1 - - -

BA - 4 - 1-4 0.25 0.3

sucrose 30,000 30,000 15,000 40,000 20,000 30,000

Phytagel 2,000 2,000 - - 2,500 2,500

pH 5.6 5.6 5.6 5.6 5.6 5.6

I Berthouly & Michaux-Ferd~re (unpublished).

2 Dufour & Carasco (unpublished).

3 Dublin (1984).

4 Murashige & Skoog (1962).

uniform plant material, the procedure is of great value in accelerating classical coffee breeding methods; e.g. in the rapid multiplication of intraspecific hybrid vari- eties of C. arabica that combine multiple resistance and hybrid vigour. The recent application of biore- actors for large scale multiplication of coffee plants has further advanced these developments (Ducos et al. 1993; Noriega & S6ndahl, 1993). Besides, a well- controlled somatic embryogenesis procedure is a sup- port for genetic transformation purposes.

In general, two approaches to liquid culture for somatic embryogenesis have been used. On the one hand, when a large quantity of embryogenic callus is required, as for genetic transformation experiments, a rapid growth of embryogenic callus is desired. Alterna- tively, a slow growth rate to minimise culture handling is desirable for long-term maintenance of embryogenic cultures. Both approaches require the maintenance of

embryogenic potential and suspension quality without actual embryo formation. Similar systems have been developed in an array of species (Vasil & Vasil, 1986; Gray & Mortensen, 1987; Muralidharan et al., 1989; Nadel et al., 1989; Chee & Cantliffe, 1989).

In this study, the main objectives were to improve the yield of the present somatic embryogenesis proce- dures for coffee, and to identify culture steps applicable to genetic transformation techniques. A two-step pro- cedure on solid medium for the production of HFSE- callus from leaf explants was utilised (Berthouly & Michaux-Ferri~re, unpubl.). The efficiency of this pro- cedure was tested on a range of coffee genotypes belonging to different Coffea species. In addition, con- ditions for the proliferation and regeneration of HFSE- callus in liquid medium were described.

Materials and methods

Plant material

Induction of embryogenic callus The plant genotypes tested belonged to four different Coffea species: C. arabica varieties ('Mundo Novo', 'Caturra' and 'Catuai') and semi-wild accessions from Ethiopia (KF2.1, AR15); C. canephora clones from Togo (107, 181, 182, 197, 202), Costa Rica (T3561- 2.1, T3561-2.3), and open-pollinated progenies from Crte d'Ivoire (OP461, OP597); Arabusta (= interspe- cific hybrid of C. arabica and C. canephora) clones from Guyana (1307, 1312) and a Congusta (= inter- specific hybrid of C. congiensis Fr. and C. canephora) clone from Madagascar (HA). Healthy greenhouse- grown plants were selected as sources for leaf explants. Disinfected young leaves from orthotropic nodes were cut into pieces of 1 cm 2 and placed, upper surface down, on callus induction medium, C (Table 1). The explants were cultured in the dark at 27 °C in petri plates (OPTILUX, Falcon). After 1 month, primary callus and explants were transferred to 100 ml glass jars, containing 25 ml of embryo induction medium, E (Table 1), and cultured at PPF (2 #mol m -2 s -1 on a 12-h photoperiod).

Suspensions with embryogenic potential Within 2-4 months (depending on genotype), fri- able embryogenic callus developed on explants on E- medium. At that time, 0.25 g of callus was transferred to 25 ml of liquid callus proliferation medium, CP (Table 1), in 150 ml Erlenmeyer flasks. The resulting suspensions were cultured at 27 °C on a gyratory shak- er at 100 rpm. Stable embryogenic suspensions were obtained 2-3 months after initiation in liquid medi- um. For subculture in 250 ml Erlenmeyer flasks con- taining 60 ml of CP-medium, only aggregates below 1 mm in diameter were collected by using a narrow mouth pipet (Falcon). The tissue grew about 3- to 4-fold between each subculture. Stable embryogenic suspensions of several coffee genotypes obtained by this procedure were used for more detailed testing of liquid culture variables, including 2,4-D concentration in CP-medium (4.5 or 9/zM), photosynthetic photon flux PPF (3 or 30 #mol m -2 s- l) , subculture interval (3, 6 or 14 days) and initial culture density (0.5, 2, 5, 10 or 20 g callus 1-1 ). The last variable was tested over a culture period of 8 weeks without the density being readjusted during subculture, and, over a period of 4

weeks, by weekly readjusting its density to the initial density. Fresh weights were used as the measure of suspension growth.

Regeneration of somatic embryos Callus suspensions were directed towards embryo for- mation by transferring to a regeneration medium, R (Table 1), containing either 4.4 or 17.6 #M BA. They were cultured at low inoculum density (1, 3 or 5 g callus 1-1) in 250 ml Erlenmeyer flasks with 75 ml of medium, under low PPF (5 #mol m -2 s - l ; 12-h pho- toperiod) at 100 rpm, and subcultured once a month. Eight to 10 weeks later, mature embryos were placed with shoot apex down on solidified medium EG (Table 1) for embryo germination. Earlier experiments had shown higher embryo germination rates in this config- uration, rather than in the usual position with root apex down.

Development into plantlets After 6--8 weeks growth on EG-medium, plantlets had developed a first pair of true leaves. At this time, cotyledons and roots were cut off and the plantlets were transferred to DEV-medium (Table 1) for development. By the time that the plantlets were 3--4 cm tall and pos- sessed 4 pairs of leaves, the in vitro-developed root was again removed and the plants were immersed in an IBA/IAA-containing solution for approximately 12 h to enhance root formation. Plants were individually potted in a soil mixture and hardened in the greenhouse during a 3-week period under a plastic cover.

Absorption of nutrients

The concentration of nutrients in CP-medium during culture was determined after dilution in lanthanum oxide, by atomic absorption spectrometry (PERKIN- ELMER 2380) (Anonymous, 1992). Analysis of nitro- gen sources was performed by the Kjeldahl method (Vogel, 1961).

Data analysis

For all data, analyses of variance using Type III sums of squares generated from the General Linear Model procedure in SAS were performed. Differences among treatment means were analyzed by Newman-Keuls test (Bliss, 1967). Differences significant at 5% probability level were considered meaningful.

10

Table 2. LFSE-embryo and HFSE-callus formation on leaf expla"ts, expressed in % of responsive expla"ts, of various Coffea genotypes after 1 month on C-medium and 75 days subsequent culture on E-medium.

Genotypes Total number Expla"ts with nodular At day 105 of expla"ts callus at day 60 (%)

Polyphenolic Expla-ts with Expla"ts with oxidation and LFSE-embryo friable I-IFSE- nodular callus formation (%) callus formation degeneration 1 (%)

C. arabica cv Mundo Novo 32 91 ... cv Catuai 33 100 ... KF 2.1 55 89 ...AR 15 20 90

C. canephora clone 107 (C x G) 2 46 17 ...... 181(C x G) 33 91 ...... 182 (C) 82 61 ...... 197 (G) 47 0 ...... 202 56 100 ...... OP 3 461 (C × G) 60 95 ...... OP 597 (C) 20 100 ... clone T3561-2.1 (C) 59 83 ...... T3561-2.3 (C) 97 100

+ 5.1 0.0 + 8.0 5.7 ++ 10.2 10.0 + 2.0 0.0

+ 0 1.0 + 7.6 90.9 ++ 0 0.5 + 0 0.0 +++ 0 66.1 + 0 82.0 + 0 25.0 ++ 3.4 35.6 + 9.3 96.9

Arabusta clone 1307 40 100 +4-+ 0 0.0 ...... 1312 53 100 + 0 66.0

Congusta clone HA 20 100 ++ 0 60.0

1 +4-4- extensive, ++ moderate, + slight. 2C: Congolese type; G: Guinea- type; C x G: F1 Hybrid. 30P, open pollinated progeny.

R e s u l t s

Induction of high frequency embryogenic callus from leaves on solid culture medium

The medium sequence C + E was initially developed

at CATIE (Costa Rica) for HFSE-callus formation in

C. canephora cultures. We tested the sequence on

a range of genotypes belonging to different species (Table 2). Development of a "nodular" callus seemed to be required for subsequent HFSE-callus formation. C. canephora clones 107 and 197 showed few nodular calli and practically no HFSE-callus formation. Other genotypes like C. arabica 'Mundo Novo' and AR15 showed exclusively the formation of low frequency somatic embryogenesis (LFSE). LFSE results when somatic embryos develop rapidly from compact nodu- lar callus. The histological characterization of such a

process was first described by S6ndahl et al. (1979) and later confirmed by Michaux-Ferri~re et al. (1989).

In our study, the first friable embryogenic HFSE-callus on leaves of C. arabica 'Catuai ' and KF2.1 appeared after some 4 months on E-medium (data not shown).

This time lag is in accordance with results of SOndahl

et al. (1985) obtained on a larger range of C. arabica varieties. Therefore their procedure might be prefer-

able for HFSE on C. arabica species. Although nodular callus had developed on the

explants, C. canephora clone 182 and Arabusta clone 1307 produced very little embryogenic callus (LFSE or HFSE) (Table 2). The best HFSE response was seen with C. canephora (181,202, T3561-2.3 and OP461), Arabusta (1312) and Congusta (HA). The formation of HFSE-callus was not always hindered by severe polyphenolic oxidation of tissue, as for example in clone 202, but, in general, a better I-IFSE-response

11

was obtained where polyphenolic oxidation occurred later in the culture cycle.

A larger variability in I-IFSE-response was observed among C. canephora genotypes than among genotypes of C. arabica. A higher level of diversity may be found in C. canephora germplasm due to its self-incompatible character and might be a reason for the generally higher capacity for regeneration of its tissue (S6ndahl et al., 1981). Looking at the genetic background of the C. canephora clones reveals differ- ences in origin (Congolese or Guinean types) but no general rule emerges to explain the observed differ- ences in tissue response (Table 2).

Proliferation of embryogenic tissue in liquid culture medium

HFSE-callus obtained after application of media sequence C + E was used to test several culture con- ditions in liquid medium. The basal medium is CP- medium (Table 1), which was developed for the pro- liferation of coffee callus in suspension cultures.

Effect of 2,4-D concentration No significant effect of altered 2,4-D concentration (described in Materials and methods) was observed (Fig. la), Thus, 4.5 #M 2,4-D and 4.6 #M kinetin were chosen for further testing of liquid culture condi- tions.

fresh weight (g I - ')

I I 4.5 plVI 2,~,-D

250 " "

2OO

150

I oo

5O

0 0 2 4 6 8 10

culture time (weeks)

b fresh weight (g I " )

5°olin 3pmol m'~s -' 30 pmo l m "~ s "~

400

3OO

ZOO

tO0

0 0 1 2 3 4 5 6 7 8 9 10

culture time (weeks)

Effect of photosynthetic photon flux Compared to growth in subdued light conditions, tis- sue growth was inhibited under intensive PPF (Fig. lb). Higher PPF may be used for long-term mainte- nance due to its growth retarding effect, although, the risk of polyphenolic oxidation in light would limit this application (Monaco et al., 1977). Growth under low PPF slowed after 9 weeks of culture (Fig. lb), when a growth plateau seemed to be achieved. Callus den- sities higher than 400 g fresh weight 1-1 of culture medium were never obtained without cell death or loss of embryogenic potential.

Effect of different subculture intervals Less tissue proliferation was observed when it was subcultured at 3-day intervals rather than at 6- or 14- day intervals (Fig. lc). No difference was observed between the 6- and 14-day subculture intervals. To minimise culture handling without adversely influenc-

C fresh weight (g I-' } 200j

150

100

SO

mm 3 days 223 6 days I'-'1 14 days

0 2 4 6 8

culture t ime (weeks)

Fig. 1. Effect of (a) 2,4-D concentration (with PPF 3 #tool m -2 s - I ; subcultufing every 2 weeks), fo) PPF (without subculturing) and (c) subculture frequency, on growth expressed in fresh weight of embryogenic callus cultures of C. canephora clone 3561(2.3) in CP-medium with an inoculum density of 6 g 1-1.

12

250

200

150

100

50

fresh weight (g 1"41

0.5g l "~ ,)K -4- 2 g l " /

"-IK- 5 g l " / / + l O g l "~ ~ /

-)~ 20 g I "~ J, ~______~ ~ , X ~ / T M

0 1 2 '~ 4 5 6 7 8

culture time (weeks)

Fig. 2, Effect of inocuhm density on growth expressed in fresh weight of embryognnic callus cultures of C. canephora OP-597 in CP-medium (,I. subculturing without density readjustment).

ing suspension growth, a 14-day subculture interval was acceptable for rapid growth of tissue.

Effect of culture density Figure 2 shows the growth curves of (7. canepho- ra OP597 suspensions that were subcultured without readjustment to their initial density. Growth of 20 g 1-1 and 10 g 1-1 suspensions slowed down between weeks 6 and 8. In 0.5 g 1-1 cultures, and, to a lesser extent, in 2 g 1-t cultures, embryos were observed, whereas no embryo formation occurred in 5, I0 and 20 g 1-1 cultures. Thus, a minimal culture density of 5 g 1-1 seems to be necessary for avoiding embryo formation. Calculation of multiplication rates (MR) demonstrated an exponential growth during the first week of cul- ture for each tested culture density. MRs for 0.5, 2, 5, 10 and 20 g 1-1 cultures during this period were respectively 16.6, 5.2, 2.9, 2.8 and 2.5. During the second week, MRs of all cultures decreased to a level between 1.0 and 1.8. Thus, by culturing small amounts of embryogenic callus at a density of 5 g 1-1, a rela- tively low MR can be maintained over several weeks. Under these conditions, subculturing in order to avoid a growth plateau is less often necessary,

When cultures had been subcultured weekly with a readjustment to their initial density of 10, 15 or 20 g 1-1, MRs over a *week culture period were 2.0 -4- 0.6, 1.8 • 0.1 and 1.5 + 0.4, respectively. The MR of 10 g 1- I cultures was significantly higher than the MR of 20 g 1-1 cultures. Thus, weekly readjustment of culture density to 10 g callus 1 - t can be used for accelerating growth of callus suspensions.

a

g

oo

"6

"6

C

o =

8

".-E._

"6

100 * " ~ g ~ e

8o, ~ ~ ~ 2 0 -

O ~ I-" I I t I I I t 1 ~ - ' 1

0 2 4 6 8 I0 12 14 16 18 20

801 ~'~ " ~ . "

~ o :

40 I a""~" a 20 ~ ' ~ - o

0~ | I l i I I i I I I I

0 2 4 6 8 10 12 14 16 18 20

100 -~ 'I"~° X

60 -. ~ =

4 0 -

20 -

O- I l i ~-- i i

0 2 4 6 8 I0 12

culture time (days)

Fig. 3. Absorption of nutrients by embryogenie callus cultures of C. canephora clone 3561(2.3) in CP-medium (inoculum density 5 g 1-1; without subcnituring), Concentrations of remaining nutrients were analysed every 2 days by Kjeldahl method or atomic absorption spectrometry, and data are given in percentages of initial concentra- tions in liquid medium. (a) nitrogen sources: Nt, total nitrogen; NO3, nitrate; NI~, ammonium. Co) macrominerals: Ca, calcium; K, potassium; Mg, magnesium; P, phosphorus. (c) microminerals:/Van, manganese; Zn, zinc.

Absorption of nutrients by embryogenic callus Absorption of some macro- and micro-salts by embryogenic suspensions was followed by regularly analysing the amount of remaining nutrients in the cul- ture medium. After 12 days of culture, 60 to 70% of the initial concentration of nutrients was still present (Fig.

[ 0 embryo formation

[moculum oensl~y (g/IJ I

Fig. 4. Effect of BA concentration (1 or4mg l -l) and culture den- sity (1, 3 or 5 g callus 1- l ) on growth, expressed in fresh weight, and embryo formation of embryogenic callus cultures of C. canephora clone T3561(2.3) in R-medium.

3). After 3 weeks, macro-salts and nitrogen sources had decreased by 50-70%, from which the amount of avail- able magnesium was lowest (30% of initial concentra- tion). These results suggest that supply of MS macro- and micro-minerals at half strength, as in CP-medium, does not lead to their depletion in embryogenic coffee suspensions, for at least 3 weeks of culture.

In trying to optimize long-term maintenance of embryogenic cultures, instead of subculturing in a completely fresh medium every 2 weeks, a part of the embryogenic callus was discarded and nutrients in concentrated form were added to the conditioned culture. In all cases, growth was nevertheless inhibit- ed after 2 months of culture. A subculture consisting of a complete or partial renewal of the culture medium could thus not be replaced by a simple addition of nutri- ents in concentrated form. Therefore, medium renewal remains necessary at regular time intervals in order to sustain the growth of embryogenic suspensions, pre- sumably by eliminating growth-inhibiting substances present in conditioned medium.

Maintenance of embryogenic capacity The embryogenic potential of suspension cultures of different genotypes was verified regularly by cultur- ing a small amount of tissue in R-medium. Except for 'Catuai' and 'Caturra', C. arabica callus was difficult to maintain in an "undifferentiated" state. 'Catuai' and 'Caturra' lost their embryogenic potential after only 16-18 months of culture in CP-medium. By this time, aggregates had gradually changed in size (< 250 #m), colour (white), and texture (spongy), with very large

13

vacuolated cells. With 'Mundo Novo', KF2.1, and ARI5, it was not possible to suppress a shift towards somatic embryo formation after 4-6 months of culture in CP-medium. This result might be due to the type of callus used by these 3 genotypes for suspension initi- ation. This callus was not friable but rather a nodular LFSE-Iike callus.

The highly embryogenic callus of clone 197 (induced on auxin-free medium) could also not be maintained in an "undifferentiated" state in CP- medium; all aggregates had developed into embryos after 7 months. Cultures of clones T3561-2.1 and T3561-2.3 preserved embryogenic capacity during 13 and 16 months of cultivation, respectively. Callus sus- pensions of OP597 were still embryogenic after 24 months of culture.

The aggregate size that was most appropriate for proliferation was between 250 and 1000 #m. The cal- lus fraction below 250 #m contained small aggregates with fewer embryogenic cells and therefore risks loos- ing its embryogenic character during sustained culture. Experiments, carried out with the fraction below 100 #m (1-10 cells per aggregate), showed a rapid degen- eration of cells or formation of non-embryogenic free cells.

Somatic embryo development in liquid culture medium

Effect of BA and inoculum density Figure 4 summarizes the effect of benzyladenine (BA) concentration and inoculum density on the occurrence of globular somatic embryos, and the increase in cal- lus fresh weight in R-medium for C. canephora clone T3561-2.3. The occurrence of globular embryos was inversely related to the inoculum density. Most early and abundant somatic embryo formation was observed with inoculum density of 1 g 1-1 combined with BA concentration of 4.4 #M. Zamarripa (1993) demon- strated the presence of inhibitory substances of somatic embryogenesis in coffee callus cultured at high density. In our experiments, somatic embryos were only formed at relatively high inoculum density (3 g 1-1) when BA concentration was increased (17.6 #M). Hence, addi- tion of BA appears to compensate for the presence of inhibitory substances to allow embryo formation to occur.

14

Table 3. Efficiency rates of coffee micropropagation systems by somatic embryogenesis in liquid culture.

Authors Species Culture method Multiplication rate Number of somatic (growth/week) embryos per

gram inoculum

Somatic embryos that developed into greenhouse plants (%)

Staritsky & van Hasselt (1980) C. canephora Flask 1.19 500-1000 40--60 Neuenschwander & Baumann C. arabica Flask 1.48 609 - (1992) Zamarripa (1993) C. canephora Flask 1.36-1.52 200 000-500 000 48 ... Arabusta . . . . . . . . . 44 Noriega & SOndahl (1993) C. arabica Bioreactor - 12 500 - Ducos et al. (1993) C. canephora Bioreactor - 600 000 47 ... Arabusta ... - . .. 35 present paper C. canephora Flask 1.81 120 000 - ... Arabusta ... - 92 300 - ... C. arabica ... 1.54 12 300 -

- not evaluated.

Conversion rates The somatic embryogenesis procedure for mass propa- gation, describedin this paper, takes about 7-8 months for C. canephora and Arabusta, and 9-10 months for C. arabica from leaf explant to regenerant (Fig. 5). The efficiencies of somatic embryogenesis procedures in liquid medium are summarized in Table 3. C. ara-

bica appears more recalcitrant to the reported proce- dures. The level of somatic embryo production is far below that of C. canephora and Arabusta. Zamarri- pa (1993) obtained relatively high rates of 200,000- 500,000 somatic embryos from 1 g of embryogenic C. canephora tissue using Erlenmeyer flasks. The 8- month period that he needed for the establishment of suspension cultures could be reduced to 2-3 months in our procedure. However, somatic embryo production was also reduced to one quarter of that in Zamarripa's procedure (Table 3).

D i s c u s s i o n

Somatic embryogenesis of coffee has been obtained by single-step (Dublin, 1981; Yasuda et al., 1985; Hatana- ka et al., 1991) and double-step procedures (S6ndahl & Sharp, 1977; Dublin, 1984; Zamarripa et al., 1991; Neuenschwander & Baumann, 1992). The single-step procedure possesses the advantage of rapid embryo formation, high embryo germination rate, and may function on genotypes recalcitrant for HFSE. HFSE, which arises from the double-step procedure, has clear

implications with regard to large scale production of somatic embryos. In addition, for some genotypes, the HFSE process may be the sole procedure for obtaining somatic embryogenesis.

In this report, we have described an HFSE proce- dure applied successfully to leaves of C. canephora,

Arabusta, Congusta and C. arabica. It appeared that response to the HFSE procedure is determined by fac- tors including genotype and occurrence of polypheno- lic oxidation. In general, lower levels of polypheno- lic oxidation in the tissue were favourable for subse- quent HFSE-callus formation. HFSE-response differed between and within species, with C. arabica being less reactive, and C. canephora and Arabusta clones more variable in their response. S6ndahl et al. (1981) showed the possibility of adapting the composition of the callus induction medium for obtaining HFSE with eight different Coffea species. Bieysse et al. (1993) also reported such adaptations for LFSE with eight different C. arabica genotypes.

A blockage in embryogenic callus formation was observed with recalcitrant genotypes ('Mundo Novo', AR15, clone 182 and clone 1307). Preliminary exper- iments, using high pressure liquid chromatography (HPLC) to determine endogenous hormone levels in callus developed on C-medium, indicated that the aux- in/cytokinin ratio at day 30 in non-embryogenic callus of a recalcitrant genotype was 3-fold higher than the ratio in callus of a relatively well-reacting genotype. We have seen that controlled lowering of the ratio at day 30 by transfer of explants to E-medium, permits

15

Fig. 5. Highfrequencys•maticembry•genesisinC.caneph•ragen•types.(a)HFSE-ca••us•n•P597•eafexp•antafter•5weeks•nE-medium (bar = 5 mm); (b) embryogenic callus of clone 181 after 3 months in CP-medium (bar = 0.5 nun); (c) regenerating embryogenic callus of clone 3561(2.3) after 9 weeks in R-medium (bar = 2 mm); (d) maturation of somatic embryos of clone 197 after 10 weeks in R-medium (bar = 8 mm); (e) fully developed plantlet of C. arabica cv. Catuai after 2 months on EG-medium (bar = 3 mm).

the development of HFSE-callus on the latter geno- type. It was reported that in order to obtain the optimal response for somatic embryogenesis, the callus must be transferred to embryo induction medium when it contains a great number of typically pre-embryogenic cells (Michaux-Ferri&e & Schwendiman, 1992). An increase of cytokinins or reduction of auxins in C- medium, combined with early transfer to E-medium, may support the appearance of such cells with recalci- trant genotypes. Further studies are needed to confirm this hypothesis.

Optimal culture density was studied with the dual objective of defining conditions for slow growth for long-term maintenance of embryogenic potential and for identifying conditions for rapid growth of embryo- genic tissue. In both cases, embryogenic potential and suspension quality should be maintained without the

occurrence of embryo formation. For the first objec- tive, an initial density of 5-10 g fresh weight 1-1 was optimal with a monthly renewal of medium. Part of the conditioned medium may be retained, depending on the quality and character of culture. PPF during culture seemed to be of less importance, but in order to avoid oxidation of polyphenols, low PPF is recommended. This way, embryogenic potential of C. canephora sus- pensions could be maintained for at least 24 months. On the other hand, when a large quantity of tissue is needed, rapid suspension growth is desired. An initial density of 10-15 g 1 - l was optimal with a complete renewal of medium every 10 days and readjusted cul- ture density of 10-15 g 1-1. PPF during such culture should preferably be low, and for both objectives tis- sue aggregates of 250-1000 #m in size appeared to

16

be optimal for nutrient absorption and maintenance of embryogenic potential.

We have shown that embryogenic suspensions could be directed towards embryo formation and sub- sequent ontogenesis by omitting 2,4-D and adding BA. This process was further enhanced by lowering the cul- ture inoculum density (1 g 1-1) and increasing the PPF (5-10 #mol m -2 s - l ) .

This somatic embryogenesis procedure for mass propagation takes about 7-8 months for C. canepho-

ra and Arabusta, and 9-10 months for C. arabica,

from leaf explant to regenerated plantlet. The prolif- eration phase in liquid medium can be prolonged at will when higher production of embryogenic aggre- gates is required. Therefore, we think that this proce- dure may be of potential interest for providing large quantities of embryogenic tissue for genetic transfor- mation purposes. The gain in time (6 months) between this procedure and the somatic embryogenesis proce- dure described by Zamarr ipa et al. (1991) is due to

the difference in time needed for culture establishment in liquid medium. In their procedure, callus originates from secondary culture on embryo induction medium that was previously described by Yasuda et al. (1985). Such callus is of less abundant and less friable character than the HFSE-cal lus described in our procedure. For this reason, optimal suspension quality in our hands could already be achieved 6 to 8 weeks after culture

initiation in CP-medium. Embryo maturation and conversion into plantlets

are factors that might still be improved in the HFSE procedure. Michaux-Ferri~re & Schwendiman (1992) reported that too rapid somatic embryo formation could induce abnormalit ies (low protein reserves, absence of shoot apices) due to embryo immaturity. Somat- ic embryo conversion rates should be optimized by a reconsideration of embryo maturation, especially the desiccation and dormancy process. Currently we are

trying to adapt our somatic embryogenesis procedure to the SIT-system (Syst~me en Immersion Temporaire) as described by Alvard et al. (1993). This system may permit HFSE from callus induction to plantlet regen- eration in one and the same culture vessel, in which optimal conditions for embryo maturation can be well- controlled.

Acknowledgements

The authors thank S. Assemat, C. Carasco and the team of CATIE, Costa Rica, for their technical assis-

tance. We are grateful to M. Dufour, A.B. Eskes and N. Michaux-Ferri~re for valuable support of this study.

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