PlantCell Reports

Plant Cell Reports (1991) 10:102 105

:9 Springer-Verlag1991

Somatic embryogenesis from leaf protoplasts of Rauvolfia vomitoria shoot cultures Jocelyne Tr~mouillaux-Guiller and Jean-Claude Ch~nieux Laboratoire de Biologie Cellulaire et Biochimie V~g6tale, Facult~ de Pharmacie, 2bis Boulevard Tonnell~, 37042 Tours c6dex, France Received February 12, 1991/Revised version received March 14, 1991 - Communicated by I. Potrykus

Abstract

Rauvolfia vomitoria mesophyll protoplasts have been isolated from axenic shoot cultures and cultured (105-106 protoplasts per ml) in Murashige and Tucker liquid medium containing growth regulators. Within 6-8 weeks, a mixed population of calli and proembryos were obtained and transferred on solid media. Calli produced shoots ; however, rooting did not occur. Somatic embryos achieved different patterns of development. In particular, whole plantlets have been obtained either directly through germination of primary embryos or via embryogenic calli. Abbreviations

BS, Gamborg et aL (1968) medium ; BA, N6 (benzyl) adenine ; 2,4-D, 2,4-dichlorophenoxyacetic acid ; MT, Murashige and Tucker (1969) medium; NAA, naphthalene acetic acid ; Z, zeatin ; K, kinetin. Introduction

Rauvolfia vomitoria Afz (Apocynaceae) is a small tree in Tropical Africa, either growing naturally or being cultivated.Chemical investigation of the roots, leafed stems and fruits showed the presence of several types of indole alkaloids [Iwu and Court 1977 ; Iwu 1980 and references therein]. The species plays an important role in pharmacology. The weakly basic fraction and reserpine, isolated mainly from the roots and stems, reduces hypertension ; they are also used as an adjunct in psychiatry [Iwu 1980]. Extracts of roots, leaves or stems are also largely used for the treatment of several diseases in African traditional medicine [Adjanohoun

1988]. Protoplast culture could be an appropriate technology for the genetic improvement of R. vomitoria. Unfortunately, regenerating plants from protoplasts of woody species remains a complex problem, due to the low digestibility of mesophyll or to the low viability of protoplasts. In this paper, we describe the culture of Offprint requests to: J.-C. Ch6nieux

mesophyll protoplasts isolated from multiple shoot cultures of R. vomitoria, together with the development of somatic embryos into plantlets. Materials and methods

Plant material In September 1985, we prepared a callus culture of Rauvolfia vomitoria Afz from a seedling root explant placed on B5 agar medium supplemented with 4.5 #M 2,4-D, 0.28 ~M K and 0.17 M glucose (pH 5.7). In November 1986, pieces of callus were transferred on MT agar medium supplemented with 4.4 p.M BA and 0.17 M glucose. Multiple shoots developing on the tissues were subcultured at 4-week intervals and maintained on the latter medium under continuous white light (Philips TL/40 W34 fluorescent tubes, 1000 lux) at 24 ~ _+1~ Protoolast isolation Two sets of experiments were performed. Protoplasts were prepared either from the tiny leaves (1-3 mm length) on the shoots developed on the above-mentioned callus culture (first set of experiments) or from the more developed leaves (10 mm length) excised from a protoplast-derived shoot culture (second set of experiments). In this latter case, the leaves were cut into strips, 0.5-0.8 mm wide. Approximately 0.5g of leaf material was incubated in 4 ml of enzyme solution (in 55 x 15 mm Petri dishes) for 14-16h, in the dark. The enzyme mixture consisted of 2.5g/I cellulase "Onozuka" R10, 1.25g/I macerozyme R10 (Yakult Honsha Co., Ltd, Japan), 0.25g/I pectolyase (Sigma-Chimie, France) in 34 mM CaCI 2 2H20 and 0.6 M mannitol (pH 5.5). After digestion, protoplasts were filtered through a 100 mm sieve and collected by centrifugation (80 x g) for 3 rain. Supernatants were discarded and pellets resuspended in 0.7 M mannitol. After centrifugation (80 x g, 3 min), protoplasts were collected in the pellet and washed again twice in 0.7 M mannitol. Following washings, protoplasts

103 content medium G/4. No matter which medium was used, protoplasts were isolated with goad reproducibility without having to strip the lower epidermis of the leaves.

were resuspended in the liquid medium MT1 (see Table I for the formulation of the media). Viability of protoplasts was checked using the induction of fluorescence in living cells with a lg/I fluorescein diacetate solution in acetone for 1-5 min : about 20 % of the preparation consisted of broken protoplasts and cellular debris, but the survival percentage of the living protoplasts reached 95 % after two days of culture.

Direct embryoq_ enesis,

Protoplasts were cultured in liquid media and similar results were obtained in the two sets of experiments. Cell walls, indicated by shape changes of the cells, were regenerated after 24-48h. About 25 % o f protoplasts

Table 1. Composition of media used in the present work MT1 MT2 MT3 MT4 MT MTBA MT60 MT60Z

G/4

MT120Z

(M) Glucose 0.25 0.25 0.16 0.16 0.16 0.16 Mannitol 0.35 0.15 0.05 Sucrose

0.04 0,17

0.17

9

9

0.34

(g/I) Agar (~M) 2,4-D NAA BA K Z

(9)*

4.5 5.4 0.4 0.5

4.5 2.3 5.4 2.7 0.4 0,2 0.5 0.25

2.3 2.7 0,2 0,25

9

9

9

9

4.5 4.5

basal medium = mineral salts and organic addition of MT medium. (*) MT4 was used either as liquid medium or as agar medium Protoplast culture.

Protoplasts were cultured at concentrations either of 105 (1st set of experiments) or of 106 (2nd set of experiments) per ml, in 35 mm Coming dishes containing 0.5 ml medium MT1. They were initially kept in the dark, at 25 + 1~ After two weeks (1st experiments) or one week (2nd experiments), the cultures were diluted with 0.2 ml medium MT2, and placed under white light (Philips TDL 18 W/33 fluorescent tubes, 750 lux) with a 12h photoperiod. Two weeks later, developing cultures were diluted at weekly intervals with 0.2-0.4 ml medium. The first feeding was done with liquid medium MT2, the second with MT3 and the third with MT4. The cultures were subdivided into two Coming dishes when the liquid culture medium exceeded 1 ml. Within one month after the third feeding, protoplast-derivated cells gave rise to a mixed population of microcalli and proembryos. Microcalli (1-2 mm in diameter) and proembryos were plated onto an agar medium, MT4. and transferred after four weeks onto the various media to be investigated. Results and discussion

In the first set of experiments, protoplasts were isolated from leaves of R. vomitoria axenic shoots which had developed from a callus, after transferring it from a 2,4-Dsupplemented B5 medium to a 2,4-D-free and BAsupplemented MT-medium. The shoots were propagated by routine periodic subcultures, and they had very small leaves (Fig 1A). In the second set of experiments, protoplasts were isolated from leaves of axenic shoots which were regenerated from a prctoplast-derived callus obtained during the first set of experiments ; the latter shoots, bearing well-developed leaves (Fig. 1B), were propagated on the hormone-free and low-glucose

4.5

Fig. 1 Shoots used for isolating protoplasts. A : shoots developed on root explant-derived callus. B : protoplast-derived shoot culture divided within 5 days (Fig. 2A-B). After 4-6 weeks, the cultures contained two types of cells : one type was present in colonies of 10-15 slightly chlorophyllous celts ; the second one was present as isolated isodiametric cells with a granular light yellow cytoplasm, a characteristic which is usually found in embryogenic cells [Song et ai. 1990]. The colonies of cells progressively increased in size, giving green microcalli (Fig.2C), whereas the isolated cells gave rise to proembryos together with some elongated cells (suspensor-like structures : Fig.2D). The proembryos turned into globular embryos, with a central core of tightly-packed chlorophyllous cells confined within an epidermic structure ( Fig.2, E-F) ; some of them slightly elongated being similar to the "oblong embryos" described by Schiavone and Cooke (1985). We usually obtained about 80 % embryos and 20 % caili in three-month old cultures. However, in a few experiments in which we did not dilute the initial culture medium, we obtained a population of calli only; it is known that the relative percentage of embryos and calli obtained from protoplasts may be affected by the composition and dilution of the culture medium (Kobayashi et aL 1985 ; Miura and Tabata 1986 ; Song et aL 1990). The somatic embryos did not develop further in liquid media and progressively turned brown. However, they could stay alive for several months without apparent necrosis. Shoot induction

Microcalli were transferred onto agar medium MT4. They were cultured for one month before being transferred again onto medium MTBA. Shoot primordia arose on the calli within 3-5 weeks. Shoots over 1 cm (Fig. 2K) were excised and individually transferred on several media used as rooting-inducers by several authors

104

Fig. 2. Shoot regeneration and embryo development from R. vomitoria leaf protoplasts. A-B : cultured protoplasts undergoing first divisions (4-6 days in culture) ; C : cell colonies after one month ; D : Two somatic proembryos (PE) with cell structures resembling suspensor (CS) after 45 days in culture ; E-F : somatic embryos (E) presenting a spherical epidermic structure (ES) ; G : embryo-derived embryogenic calli ; H : embryo-derived rhizogenic calli , I-J : cotyledonary stage ; K : shoots developed on microcalli ; L : whole plantlet ; M : nodular calli. Bar = 50 # M (A-B) ; IO0 #M (C-F) ; I mm (I) ; 1.5 mm (J,L) ; 2 mm (G) ; 5 mm (H,K,M) [Agrawal et al. 1989 ; Kaul 1987] . However, root formation was never observed. As frequently observed with woody plants, the rooting process in R. vomitoria cultures remains obviously a complicated problem [Rastushnyak et aL 1989 ; Ochatt and Caso 1986]. On the other hand, shoots cultured on medium G/4 rapidly elongated, branched out and developed normal leaves. Development of emb~os Direct somatic embryos were transferred onto MT agar medium. About ten per cent of them swelled within one month of culture. Moreover, ten per cent of the remaining "recalcitrant" embryos also developed during the next months. The swollen embryos were cultured on solid MT medium supplemented either with a cytokinin (4.5yM BA or Z) or with a high level of sucrose (MT60). On these media different patterns of development were observed (Fig. 3). A few embryos (about one per cent) developed into whole plantlets (Fig. 2L) but some of

I Caulogenic callus I

P I cultures Shoot

I "'...... I Oo'o'ooary ",l stages

I

Protoplasts !

] a'

'i

I

I

s J Embryogenic ,,,," Icallus

I

Somatic embryos

Rhizogenic

~

Who,a

t'--'~"~'l Plantlets

- _

J\!ca,,u.

I

.,. Recallusing

~1 Noclularcallus

Fig. 3 Morphogenetic response found in cultures - - ~

R. vomitoria protoplast

Direct embryogenesis pathway Indirectembryogenesis pathway

105 them stopped their growth at the cotyledonary stage (Fig. 2 I-J), and recallused into caulogenic calli. Most of the embryos recallused into three types of calli : (a) yellowish embryogenic calli (Fig. 2G) giving rise continuously to indirect somatic embryos ; (b) white rhizogenic calli (Fig. 2H) leading to roots which rapidly stopped their development ; (c) nodular cain presenting a chlorophyllous core (Fig. 2M) ; these nodules appeared to be somewhat similar to the small sugarcane cain bearing green meristems described by Chen etal. (1988) ; upon transferring them onto different media (MT, MT60, MT60Z), they could revert to the embryogenic, rhizogenic or caulogenic cain mentioned above, the percentage for "each type depending on the composition of the medium (Table 2). In these experiments, the number of plantlets obtained either through germination of direct somatic embryos or via embryogenic callogenesis, remained very low (Table 3). We attempted to enhance the percentage of plantlet formation through increasing the osmolarity of the medium during one passage, since some work has indicated that dessication may be an important part of the maturation process (Finkelstein and Crouch 1986 ; Lu and Thorpe 1987) : as shown in Table 3, this treatment improved the production of whole plantlets. As far as we know, this study provides the first example of R. vomitoria protoplast culture. Whereas somatic embryogenesis was achieved less frequently than other methods of regeneration in woody species, R. vomitoria protoplasts can regenerate into plantlets, some of them being obtained through direct embryogenesis. As previously found by Davey and Power (1988), isolating protoplasts from shoot cultures (which can be considered as juvenile tissues) may overcome the problems which are inherent to woody species. Moreover, leafy shoots obtained from microcalli or somatic embryos provide a genetically homogenous source of protoplasts all year around. These protoplasts might be used for genetic engineering such as genome transformation and somatic hybridization. We are still trying to increase the frequency of plantlet formation from embryos and to find suitable culture conditions for growing these plantlets to mature plants. References

Adjanohoun EJ (1988) Contribution aux 6tudes ethnobotaniques et floristiques en R6publique Populaire du Congo ACCT, Paris, p 99 Agrawal S, Chandra N, Kothari SL (1989) Plant Cell Tissue Organ culture 16:47-55 Chen WH, Davey MR, Power JB, Cocking EC (1988) J. Exp. Bot. 199:251-261 Davey MR, Power JB (1988) In :Puite KJ, Dons JJM, Huizuing HJ, Kool AJ, Koornnef M, Krens FA (eds) Progress in Plant Protoplast Research, Kluwer Acad. Publ., New York, pp 15-25

Table 2. Morphogenetic response obtained with nodules growning on different media

Responses (%)

MT

Embryogenic catli

10

Medium MT60

MT60Z

12

22

30

25

34

24.5

24

Cotyledonary stage

6

4

11

Whole plantlets

0

0,9

3

Rhizogenic calli

2.5

Nodular calli

Shoots

10

13

15

No reponse

37.5

15.6

0

Total : 875 nodules. Response after 4 weeks in culture. See table I for the formulation of the media

Table 3. Whole plantlet formation from somatic embryos grown on different media

M'F MTBA Responses (%)

0.7

1.7

Medium MI-60

MT60Z

1.2

1.7

MT60Z (al MT 120 Z 16.4

Total : 1045 somatic embryos. Response after three months in culture. (a) Three transfers on MT6OZ, then one transfer on MT120Z See tabie 1 for the formulation of the media

Finkelstein RR, Crouch ML (1986) Plant Physiol. 81 : 907912 Gamborg OL, Miller RA, Ojima K (1968) Exp. Cell Res. 50:151-158 Iwu MM, Court WE (1977) Planta med. 32:88-99 Iwu MM (1980) Planta med. suppl. : 13-16 Kaul K (1987) Plant Cell Reports 6:5-7 Kobayashi S, Ikeda I, Uchimiya H (1985) Plant Cell Tissue Organ Culture 4:249-259 Lu CY, Thorpe TA (1987) J. Plant Physiol. 128:297-302 Miura Y, Tabata M ('1986) Plant Cell Reports 5:310-313 Murashige ET, Tucker DPH (1969) In: Chapman (ed.) First Int. Citrus Symp., Univ. Calif. ,Riverside, pp 1155-1161 Ochatt SJ, Caso OH (1986) J. Plant Physiol. 122:243-244 Rastushnyak YI, Piven NM, Rudas VA (1989) Plant Cell Tissue Organ Culture 17:183-190 Schiavone FM, Cook TJ (1985) Can. J. Bot. 63:15731578 Song J, Sorensen EL, Liang GH (1990) Plant Cell Reports 9:21-25