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In vitro culture of fennel tissues (Foeniculum vulgare Miller) from cell suspension to mature plant
Scientia Horticulturae, 22 (1984) 55--65
55
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
IN VITRO CULTURE OF FENNEL TISSUES (FOENICULUM VULGARE MILLER) FROM CELL SUSPENSION TO MATURE PLANT
G. H U N A U L T
Universit~ P. et M. Curie, UER 59, Station de Biologie v~g~tale "Armand de Richelieu", Le Haut-Buisson, CherrY, 72400 La FerrY-Bernard (France) (Accepted for publication 28 June 1983)
ABSTRACT Hunault, G., 1984. In vitro culture of Fennel tissues (Foeniculum vulgare Miller) from cell suspension to mature plant. Scientia Hortic., 22: 55--65. Callus produced by bitter Fennel explants cultivated on a modified Murashige and Skoog medium containing 2,4-D easily gave rise to cell suspensions when grown in a shaken liquid medium. These suspensions also produced embryoids. We here describe the several cultural stages necessary before the plantlets raised from these embryoids could be transferred to soil. More than 800 plants have been obtained in this way since 1980, and have all reached full maturity. However, most of them did not produce viable seeds and many died during the winter. These problems are discussed. Keywords: Foeniculum vulgare; in vitro culture.
INTRODUCTION
For several years, Fennel culture has been the subject of growing interest in France, in order to produce anethole (Desmarest, 1978). As plant tissue and organ culture are now widely used for crop propagation and selection, we undertook, within a larger breeding program of Fennel, to study the in vitro behaviour of bitter Fennel tissues (Foeniculum vulgare Miller ssp. capillaceum (Gilib.) Holmboe vat. vulgare (Miller) TheUung). Maheshwari and Gupta (1965) obtained callus and cell suspensions able to give embryoids. In a previous paper (Hunault, 1981), we described the behaviour of various explants grown in vitro and the formation of embryoids and plantlets from callus, thus corroborating the results of these authors. Stem and petiole segments easily produced embryogenic callus when cultivated on Murashige and Skoog's salt medium (1962) supplemented with vitamins, sugar and 2,4-D (2,4-dichlorophenoxyacetic acid). Embryoids also gave plantlets when grown without the auxin. Thus, Fennel tissues behave as most other embryogenic systems, and are particularly similar to other Umbelliferae such as carrot or celery. 0304-4238/84/$03.00
© 1984 Elsevier Science Publishers B.V.
56 Our aim being to obtain variants amongst the plant population raised from embryoids, we thought that treatments which might increase variability (mutagens, polyploidizing substances, etc.) would be more effective on small cell clumps than on callus, and we thus tried to get cell suspensions. As fennel plantlets withered rapidly when transferred directly from test tubes to soil~ it was necessary to develop a technical process to allow a large number of them to grow outdoors and reach full maturity. In this paper, we describe the various cultural stages that enabled us to grow numerous plantlets obtained from cell suspensions to the mature state. MATERIALS AND METHODS Primary calli were initiated from explants (stem or petiole pieces) of bitter fennel plantlets grown on a medium containing Murashige and Skoog's salts (1962) (MS), thiamine-HC1 and myo-inositol following Linsmaier and Skoog (1965), 3% glucose, 1 mg 1-1 2,4-D (Na salt), 0.3 mg 1-1 kinetin and 0.7% agar. The plantlets from which explants were taken were obtained either from aseptically grown seeds or from embryoids. Conditions for growing the seedlings and the plantlets raised from embryoids have been given previously (Hunault, 1981). Callus cultures were maintained on the same medium and sub-cultured every 6 weeks. Cell suspensions were also grown in this medium without agar. Kinetin was reduced to 0.1 mg 1-1 or omitted, and the pH was adjusted to 5.5 with 1 N and 0.1 N NaOH before autoclaving. Each suspension was obtained by transferring a whole callus into a 500 ml Erlenmeyer flask containing 250 ml of medium. These flasks were p u t on a vertical gyratory shaker and rotated at 56 r.p.m. Suspensions were sub-cultured at monthly intervals by adding 10 ml of stationary culture to 250 ml of new medium. All the cultures were exposed to continuous fluorescent light (Mazda T F R S 4 0 / L J L , 800 lux) and maintained at 25 ° C. In order to succeed in growing plants to maturity, a number of transfer steps on several slightly different media had to be developed. Details on these media will be given in the text. RESULTS S u s p e n s i o n cultures o f f e n n e l tissues. -- During this study, the development
of callus parts differing in their gross morphology was often observed. This allowed different lines of embryogenic tissue to be isolated, either at the end of the primary culture or during the following transfers. Differences between these lines were found in their colour (pale green, yellow, whitish cream or greyish) or in their texture, some of them being soft and flaccid, others being finely or coarsely granulous. Most of these granulous tissues could n o t be grown as suspensions because they then gave large nodules reaching several mm in diameter. Other callus types quickly gave cell suspensions when
57 shaken in the liquid medium. According to their size and structure, cells could be separated into two main classes: large and vacuolated cells (meristematic parenchyma cells) and small cytoplasmic cells (primary meristematic cells). Large cells were very variable in size and shape. Most of them were rounded or ovoid and 80--100 pm in diameter. However, some were very elongated, sometimes spirally, and were several hundred pm in length. These large cells were generally aggregated into loose clumps that dissociated easily to give smaller aggregates and free cells. Small cells were generally isodiametric (about 20 ~m in diameter), densely cytoplasmic, with small vacuoles and large nuclei, and often filled with numerous starch grains. They formed compact clumps, which broke up during the suspension growth giving smaller groups that continued proliferating. These small cells seldom grew free. A third cell type, consisting of cells with plastids, was observed in green strains. Similar to the first cell type described, these cells had large vacuoles but were generally smaller (60--90 pm in length) and bowed in shape. They often developed as irregular files. As they aged, their plastids became filled with large starch grains. These 3 cell types were not always spatially separated and often constituted mixed clumps. Ontogenic relations between these various cells have not been studied. As previously stated by Maheshwari and Gupta (1965), cell suspensions behave like calli and produced embryoids when they were grown without 2,4-D (Fig. 1 a--e). Most of these embryoids developed from the periphery of clumps, where they formed clusters. They then became free-floating in the medium, being isolated or joined by their basal (root) end. Squashes of aggregates taken from recently induced embryogenic cultures showed young embryoids of various shapes and structures, with the first stages being linear or rounded, as in carrot (Halperin and Jensen, 1967; Backs-Hiisemann and Reinert, 1970; Halperin, 1970; Reinert et al., 1971; McWilliam et al., 1974; Smith and Street, 1974; Street and Withers, 1974). Their origin was not studied in detail. Culture o f plantlets obtained from embryoids In a first attempt, plantlets were transferred directly from their inductive medium (agar medium without 2,4-D) to a greenhouse soil. However, most of them withered, so that a multi-stage procedure had to be developed to improve their rooting and their resistance to wilting. To obtain mature plants from cell suspension we used the following process. Stage O: Cell suspension culture (see materials and methods). Stage 1 : Induction o f embryogenesis. -- At the end of Stage 0 (1 month), 2 ml of a stationary-phase culture was pipetted into 100 ml of an inductive medium and then shaken for 12--15 days in the same way as the suspensions. The new medium consisted of half-strength MS salts, 0.4 mg 1-~ thiamineHC1 and 1.5% glucose. During this stage, embryoids began their develop-
58
59
ment. The more advanced of them reached the cotyledonary stage and acquired individuality. This was also a diluting stage, later allowing better growth of plantlets on the Stage 2 medium. It was observed that the direct transfer of the Stage 0 cell suspension, even in a small quantity, on to a solid inductive medium often further impeded plantlet development because of the presence of too many embryoids. Embryoids did not give normal plantlets when maintained in this shaken inductive medium. Although they reached 2--3 cm in length, their stem remained atrophied and they did not resume normal development when transplanted on a solid medium. However, some of them did produce new adventive embryos which grew directly into plantlets. Stage 2: Development of plantlets from embryoids. -- After 12--15 days, one or two drops of the embryoid suspension were pipetted into 26 mm X 170 mm test tubes containing about 40 ml of a solid medium. The medium was almost the same as that of Stage 1, but contained 1% powdered charcoal (Fridborg and Eriksson, 1975; Drew, 1979) and 0.7% agar. The tubes were next closed with cotton plugs but not capped because capping caused yellowing of plantlets, delay in rooting and vitrification of some stems and petioles. The liquid medium film surrounding the embryoids disappeared a few days after the transfer, and they continued their development at the agar surface. This stage was approximately 10 weeks, the first plantle~s being transplantable after 4 weeks. Many of the plantlets formed by embryoids were completely teratologic and unusable. The number of these abnormal plantlets increased as the suspension aged. Generally, a 2-year-old culture was still embryogenic but produced no more normal plantlets. Only plantlets having normal leaves were transferred. Thus, the number of these transplantable plantlets varied with the strains and with their age. For some freshly initiated lines as many as 30 plantlets per tube were obtained. However, normally their number seldom exceeded 10. In our culture conditions, plantlets had a long hypocotyl and a thin primary root. As indicated previously (Hunault, 1981), these plantlets looked almost normal but they did show some differences from normal seedlings. (i) Cotyledons were generally shorter than normal, Fig. 1. Embryoid development during the induction stage (Stage 1, medium without 2,4-D). (a, b, c) Young embryoids torn away from their producing aggregate. In most cases, a few large elongated cells of the clump core strongly stick to the basal end of each embryoid. Some of these cells have large starch grains. Smaller grains are also seen in embryoid cells. (d) Cluster of large embryoids still attached to the producing clump. Embryoids are connected to the clump by large suspensor-like cells as in (a, b, c). (e) Free floating embryoid with t w o young well-developed cotyledons. (f) Shoot part of an embryoid showing only one cotyledon with a sheathing base. (g) Embryoid with two unequal cotyledons. (h) embryoid with two equal cotyledons (as in (e), but older). During further development, such cotyledons may elongate or remain short; s o m e of them become lobate.
60
sometimes reduced to a single piece (Fig. 1, f), unequal (Fig. 1, g) or with a divided lamina. (ii) Hypocotyls sometimes coalesced, sometimes up to the cotyledonary node. (iii) Leaf laminae of the first true leaves were less dissected than normal ones, and entire in some cases. E m b r y o and plantlet malformations have previously been described for embryogenic cultures of other umbelliferous species such as carrot (Halperin and Wetherell, 1964; Homes and Guillaume, 1967), parsley (Vasil and Hildebrandt, 1966) and caraway (Ammirato, 1974), and non-umbeUiferous species such as Citrus (Button et al., 1974). Such anomalies seem inherent to m a n y systems yielding vegetative embryos. Stage 3: Increase o f rooting and resistance to wilting. ~ Plantlets having at
least one true leaf (Fig. 2, a) were isolated and their hypocotyl cut about 1 cm under the cotyledonary node (Fig. 2, b). The long hypocotyl, the primary root and the collar, which were often distorted and showed a tendency to produce new adventive embryos during sub-culture, were thus discarded. Plantlets were then grown on a liquid medium made of halfstrength MS salts, 1.5% glucose and 0.1 mg 1-1 indolebutyric acid (IBA), the remnant of the hypocotyl being inserted through the central hole of a filter-paper dome dipping into the medium. New vigorous roots generally appeared on the hypocotyl 2--3 weeks later, while the stem gave new leaves (Fig. 2, c). Some plantlets bloomed (Fig. 2, d) and set several small fruit. However, these did not reach maturity and were sterile. Such rooted plantlets m a y be transferred to Stage 4 after 4--6 weeks. Not all plantlets were suitable for transplanting because some of them did not root, while the stem of others ceased growing and often became distorted. Between 60 and 70% could be transferred, although a higher percentage was obtained when rigorous selection was done at the end of Stage 2. Plantlet vigour is diminished when glucose concentration is higher than 2%. Badoc (1982) also stated that the best glucose concentration for bitter fennel micropropagation is about 2%. Increasing the IBA concentration to 0.5 mg 1-1 increased rooting and hardiness but caused distortion and callusing of the hypocotyl base. It also promoted the production of adventive embryos from the hypocotyl and cotyledons. As these modifications of the lower parts of plantlets were detrimental to further growth, the IBA concentration, although not optimal for rooting, was maintained at 0.1 mg 1-1
Fig. 2. Morphology of plantlets during sub-cultures. (a) Plantlets with a first true leaf isolated from Stage 2 cultures. (b) Identical plantlets prepared for Stage 3 culture (lower part discarded). (c) Rooted plantlet at the end of Stage 3. (d) Flowering plantlet at the end of Stage 3. (e) Young greenhouse grown plant after 2-month culture in Stage 4; a robust offshoot is developing from a lower node and will eventually substitute for the etiolated first stem. Such a substitution often occurs during Stages 4 or 5 and generally gives hardy plants.
2 i
1 •
|
3
I
4
|
5cm
62 TABLE I Influence o f soil mixture on plantlet establishment Number of pots
Substrate
Establishment rate (%) .
120 120 120
Vermiculite Vermiculite + peat (50:50) Perlite + peat (50:50)
83 66 36
S t a g e 4: G r e e n h o u s e t r a n s p l a n t a t i o n . -- Well-developed rooted plantlets were
removed from the tubes and transplanted into pots containing a synthetic soil wetted with a commercial mineral solution (Floreden). To avoid rotting during the first week, their lower part was sprayed with a 2% oxiquinoline solution (Cryptonol). Plants were further covered with a transparent polyethylene bag and transferred to a growth room (day period: 16 h, 15 000 lux, 22°C, 70% air saturation; night period: 8 h, 18°C, 95% air saturation). A drip system allowed each pot to be individually watered with the nutrient solution. It proved very important to avoid soil saturation, because the fennel root system is highly sensitive to asphyxiation. Plastic bags were removed after 5 days. Some plants withered, but about 70% kept growing and eventually flowered. The success of establishment and subsequent plant development varied with the soil mixture (Table I). Poor results were obtained with the perlite + peat substrate. The best establishment was obtained with vermiculite alone, but growth was better on vermiculite + peat (50:50). transfer. -- Plants were transferred to the field during spring or at the beginning of summer. Leaves which had been developed during Stage 4 often wilted after transfer, but after a period of adaptation, new robust shoots appeared on the lower part of the stems. They produced numerous umbellae during the following summer and some set seeds. No particular problem arose during this transfer, the success rate being generally higher than 80%.
S t a g e 5: O u t d o o r
DISCUSSION
The process described above allows the raising of mature bitter fennel plants from suspension cultures at a relatively high success rate. The field behaviour of more than 800 plants obtained in this way have been studied since 1980. Variations occurred in the plants, indicating that they could be of value in a breeding program. However, at the present time, three obstacles impede the progress of this work. The first obstacle is the progressive loss by suspensions of their ability to produce normal plantlets. The disappearance of the embryogenic potential of various carrot cultures has been recorded by
63 several authors (Reinert, 1959; Halperin and Wetherell, 1965; Mouras and Lutz, 1973; Smith and Street, 1974) and has been related to an increase in cell sensitivity to 2,4-D and to the competitive elimination of embryogenic cells by non-competent ones, these often being aneuploid or polyploid (Smith and Street, 1974). Culture of celery tissues in a shaken liquid medium also induced the loss of their embryogenic ability (A1-Abta and Collin, 1978). After several years, our fennel strains still produce embryoids but these develop later (most of them appear during Stage 2 instead of Stage 1) and all give completely abnormal plantlets. A detailed caryological study of suspensions has not yet been done, and it may be that it is caused by a chromosomal drift. However, this transformation is probably not related to a drift toward tetraploidy because recently, a tetraploid line was isolated (most cells tetraploid with 44 chromosomes or hypotetraploid) which was able to produce a high number of morphologically normal plantlets. Tetraploid carrots were obtained in this way by Smith and Street (1974). These authors stated that the decrease in embryogenic potential was frequently correlated with the dominance of cells having chromosome numbers above the tetraploid level. The second obstacle is that plants obtained from embryoids generally exhibit a very low level of fertility. Some of them prove completely sterile, even when cultivated near a field of normal plants, i.e. in an environment permitting good open pollination. Young fennel plants begin to produce umbellae during the greenhouse culture (sometimes in the test tubes at Stage 3) and flower profusely when field grown. After the shedding of perianth pieces and stamens, ovaries start growing larger, but most of them stop developing when they reach 5--6 mm in length; they then turn yellow and later abscise. Dissection shows that fruits become yellow only after ovule shrivelling. To our knowledge, such sterility has not been quoted for carrot, but fruits of celery plants raised from embryoids do not reach maturity (Merrick and Collin, 1980). Fennel and celery are closely related plants belonging to the same tribe and having the same chromosomic number (2n = 22), while carrot is an outlying species with 18 chromosomes. Hore observed various anomalies during male meiosis of fennel (1976} and celery (1977), and asserted (1979) that the poor seed-set of various aromatic umbelliferae is the result of bad pollen quality. He also stated that fennel may present some self-incompatibility (Hore, 1979). Although cultivars used at the present time in France generally yield a high number of seeds when field grown, our in vitro culture conditions could have induced cytogenetic irregularities causing plantlets to become either completely male sterile or selfincompatible. Nevertheless, this hypothesis cannot explain the poor yield of seeds when plants are open-pollinated. Aneusomatism could be another explanation. Ogura (1976), working on tobacco plantlets regenerated from callus, and Bennici and D'Amato (1978), on wheat plantlets obtained in vitro from mesocotyl, observed that these plantlets were mixoploid and had various numbers of aneuploid cells. Moreover, Browers and Orton (1982)
64
observed a significant number of hypodiploid cells in celery plantlets regenerated from embryoids. Caryologically heterogeneous plants of tobacco were generally less fertile than normal ones (Ogura, 1976). A preliminary study of the caryological status of several plantlets produced by our tetraploid line of fennel showed that they are also mixoploid, with various numbers of hypotetraploid cells. This aneusomatism could alter both male and female meiosis, thus leading to complete sterility. A detailed study of meiosis is needed to elucidate the reason for the almost total lack of fruiting of these plants raised from embryoids. The winter death of a high number of plants is the third obstacle impeding the progress of our work. In contrast to sweet fennel (Foeniculum vulgare Mill. ssp. capillaceum (Gilib.) Holmboe var. dulce (Miller) Thellung), bitter fennel is a perennial variety. Therefore, when beginning this study, we supposed that plants, once established during summer, could be studied for several years. Thus, it was disappointing to see in spring 1982 that about 90% of plants established during the 1981 season had died during the winter. This was observed in two experimental fields about 180 km apart. It is now of interest to study if this winter sensitivity is due to peculiar aspects of our cultural process or is ascribable to deeper modifications of the properties of these plants. ACKNOWLEDGEMENTS
I am greatly indebted to P. Desmarest, Institut de Recherches Appliqu~es aux Boissons, Cr~teil, France, for his invaluable help during the greenhouse and field culture of the fennel plants. I also wish to acknowledge A. Mahuet for reading the English text.
REFERENCES A1-Abta, S. and Collin, H.A., 1978. Control of embryoid development in tissue cultures of Celery. Ann. Bot., 42: 773--782. Ammirato, P.V., 1974. The effects of abscisic acid on the development of somatic embryos from cells of caraway (Carum carvi L.). Bot. Gaz., 135: 328--337. Backs-HUsemann, D. and Reinert, J., 1970. Embryobildung durch isolierte Einzelzellen aus Gewebekulturen yon Daucus carota. Protoplasma, 70: 49--60. Badoc, A., 1982. Contribution ~ l'~tude des ph~nom~nes de morphogen~se et de callogen~se de tissus de Fenouil vulgaire ( Foeniculum vulgate ssp. capiUaceum vat. vulgare (Mill.) Thellung), analyse des constituants de l'huile essentielle des explants. DEA, Universit~ des Sciences et Techniques, L~lle, 74 pp. Bennici, A. and D'Amato, F., 1978. In vitro regeneration of Durum wheat plants. I. Chromosome numbers of regenerated plantlets. Z. Pflanzenzuecht., 81: 305--311. Browers, M.A. and Orton, T.J., 1982. Transmission of gross chromosomal variability from suspension cultures into regenerated celery plants. J. Hered., 73: 159--162. Button, J., Kochba, J. and Bornman, C.H., 1974. Fine structure of and embryoid development from embryogenic ovular callus of 'Shamouti' orange (Citrus sinensis Osb.). J. Exp. Bot., 25: 446--457.
65 Desmarest, P., 1978. N e w aspects of fennel cultivation in France. Acta Hortic., 73: 289-295. Drew, R.L.K., 1979. Effect of activated charcoal on embryogenesis and regeneration of plantlets from suspension cultures of Carrot (Daucus carota L.). Ann. Bot., 44: 387-389. Fridborg, G. and Eriksson, T., 1975. Effects of activated charcoal on growth and morphogenesis in cell cultures. Physiol. Plant., 34: 306--308. Halperin, W., 1970. Embryos from somatic plant cells. Symp. Int. Soc. Cell Biol., 9" 169--191. Halperin, W. and Jensen, W.A., 1967. Ultrastructuralchanges during growth and embryogenesis in carrot cellcultures. J. Ultrastruct. Res., 18: 428--443. Halperin, W. and Wetherell, D.F., 1964. Adventive embryony in tissue cultures of the wild carrot, Daucus carota. A m . J. Bot., 51: 274--283. Halperin, W. and Wetherell, D.F., 1965. Ontogeny of adventive embryos of wild carrot. Science, 147: 756--758. Homes, J.L.A. and Guillaume, M., 1967. Ph~nom~nes d'organogen~se dans des cultures in vitro de tissusde carotte (Daucus carota L.). Bull. Soc. R. Bot. Belg., 100: 239--258. Hore, A., 1976. Cytogenetical studies of the genus Foeniculum (Umbelliferae). Indian Agric., 20 : 183--191. Hore, A., 1977. Study of the structure and behaviour of chromosomes of the different varietiesof Apium graveolens (Celery). Cytologia, 42: 21--28. Hore, A., 1979. Improvement of minor (Umbelliferous) spices in India. Econ. Bot., 33: 290--297. Hunault, G., 1981. La culture in vitro des tissus de Fenouil (Foeniculum vulgate Miller). Premieres observations sur le c o m p o r t e m e n t des explantats primitifs et des cals. C.R. Acad. Sci. Paris, Set. III, 293: 553--558. Linsmaier, E.M. and Skoog, F., 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant., 18: 100--127. Maheshwari, S.C. and Gupta, G.R.P., 1965. Production of adventitious embryoids in vitro from stem callus of Foeniculum vulgare. Planta, 67 : 384--386. McWilliam, A.A., Smith, S.M. and Street, H.E., 1974. The origin and development of embryoids in suspension cultures of carrot (Daucus carota). Ann. Bot., 38: 243--250. Merrick, M. and Collin, H.A., 1980. Selection for resistance to the herbicide Asulam using plant tissue cultures. In: Cong. Eucarpia, Application de la culture in vitro ~ l'am~lioration des plantes potag~res, 16--18 April 1980, Versailles, France, pp. 121--125. Mouras, A. and Lutz, A., 1973. Sur les variations du comportement organog~ne des cultures de tissus de Carotte sauvage produisant des pseudo-embryons. C.R. Acad. Sci. Paris, S~r. D, 177: 1151--1153. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473--497. Ogura, H., 1976. The cytological chimeras in original regenerates from tobacco tissue cultures and in their offspring. Jpn J. Genet., 51: 161--174. Reinert, J., 1959. Uber die Kontrolle der Morphogenese und die induktion yon Adventivembryonen an Gewebekulturen aus Karotten. Planta, 53 : 318--333. Reinert, J., Backs-HUsemann, D. and Zerban, H., 1971. Determination of embryo and root formation in tissue cultures from Daucus carota. In: Coll. Int. CNRS No. 193, Les cultures de tissus de plantes, 6--10 July 1970, Strasbourg, France, pp. 261--268. Smith, S.M. and Street, H.E., 1974. The decline of embryogenic potential as callus and suspension cultures of Carrot (Daucus carota L.) are serially subcultured. Ann. Bot., 38: 223--241. Street, H.E. and Withers, L.A., 1974. The anatomy of embryogenesis in culture. In: H.Ep Street (Editor), Tissue Culture and Plant Science 1974. Academic Press, London, pp. 71--100. Vasil, I.K. and Hildebrandt, A.C., 1966. Variations of morphogenetic behavior in plant tissue cultures. II. Petroselinum hortense. Am. J. Bot., 53: 869--874.
55
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
IN VITRO CULTURE OF FENNEL TISSUES (FOENICULUM VULGARE MILLER) FROM CELL SUSPENSION TO MATURE PLANT
G. H U N A U L T
Universit~ P. et M. Curie, UER 59, Station de Biologie v~g~tale "Armand de Richelieu", Le Haut-Buisson, CherrY, 72400 La FerrY-Bernard (France) (Accepted for publication 28 June 1983)
ABSTRACT Hunault, G., 1984. In vitro culture of Fennel tissues (Foeniculum vulgare Miller) from cell suspension to mature plant. Scientia Hortic., 22: 55--65. Callus produced by bitter Fennel explants cultivated on a modified Murashige and Skoog medium containing 2,4-D easily gave rise to cell suspensions when grown in a shaken liquid medium. These suspensions also produced embryoids. We here describe the several cultural stages necessary before the plantlets raised from these embryoids could be transferred to soil. More than 800 plants have been obtained in this way since 1980, and have all reached full maturity. However, most of them did not produce viable seeds and many died during the winter. These problems are discussed. Keywords: Foeniculum vulgare; in vitro culture.
INTRODUCTION
For several years, Fennel culture has been the subject of growing interest in France, in order to produce anethole (Desmarest, 1978). As plant tissue and organ culture are now widely used for crop propagation and selection, we undertook, within a larger breeding program of Fennel, to study the in vitro behaviour of bitter Fennel tissues (Foeniculum vulgare Miller ssp. capillaceum (Gilib.) Holmboe vat. vulgare (Miller) TheUung). Maheshwari and Gupta (1965) obtained callus and cell suspensions able to give embryoids. In a previous paper (Hunault, 1981), we described the behaviour of various explants grown in vitro and the formation of embryoids and plantlets from callus, thus corroborating the results of these authors. Stem and petiole segments easily produced embryogenic callus when cultivated on Murashige and Skoog's salt medium (1962) supplemented with vitamins, sugar and 2,4-D (2,4-dichlorophenoxyacetic acid). Embryoids also gave plantlets when grown without the auxin. Thus, Fennel tissues behave as most other embryogenic systems, and are particularly similar to other Umbelliferae such as carrot or celery. 0304-4238/84/$03.00
© 1984 Elsevier Science Publishers B.V.
56 Our aim being to obtain variants amongst the plant population raised from embryoids, we thought that treatments which might increase variability (mutagens, polyploidizing substances, etc.) would be more effective on small cell clumps than on callus, and we thus tried to get cell suspensions. As fennel plantlets withered rapidly when transferred directly from test tubes to soil~ it was necessary to develop a technical process to allow a large number of them to grow outdoors and reach full maturity. In this paper, we describe the various cultural stages that enabled us to grow numerous plantlets obtained from cell suspensions to the mature state. MATERIALS AND METHODS Primary calli were initiated from explants (stem or petiole pieces) of bitter fennel plantlets grown on a medium containing Murashige and Skoog's salts (1962) (MS), thiamine-HC1 and myo-inositol following Linsmaier and Skoog (1965), 3% glucose, 1 mg 1-1 2,4-D (Na salt), 0.3 mg 1-1 kinetin and 0.7% agar. The plantlets from which explants were taken were obtained either from aseptically grown seeds or from embryoids. Conditions for growing the seedlings and the plantlets raised from embryoids have been given previously (Hunault, 1981). Callus cultures were maintained on the same medium and sub-cultured every 6 weeks. Cell suspensions were also grown in this medium without agar. Kinetin was reduced to 0.1 mg 1-1 or omitted, and the pH was adjusted to 5.5 with 1 N and 0.1 N NaOH before autoclaving. Each suspension was obtained by transferring a whole callus into a 500 ml Erlenmeyer flask containing 250 ml of medium. These flasks were p u t on a vertical gyratory shaker and rotated at 56 r.p.m. Suspensions were sub-cultured at monthly intervals by adding 10 ml of stationary culture to 250 ml of new medium. All the cultures were exposed to continuous fluorescent light (Mazda T F R S 4 0 / L J L , 800 lux) and maintained at 25 ° C. In order to succeed in growing plants to maturity, a number of transfer steps on several slightly different media had to be developed. Details on these media will be given in the text. RESULTS S u s p e n s i o n cultures o f f e n n e l tissues. -- During this study, the development
of callus parts differing in their gross morphology was often observed. This allowed different lines of embryogenic tissue to be isolated, either at the end of the primary culture or during the following transfers. Differences between these lines were found in their colour (pale green, yellow, whitish cream or greyish) or in their texture, some of them being soft and flaccid, others being finely or coarsely granulous. Most of these granulous tissues could n o t be grown as suspensions because they then gave large nodules reaching several mm in diameter. Other callus types quickly gave cell suspensions when
57 shaken in the liquid medium. According to their size and structure, cells could be separated into two main classes: large and vacuolated cells (meristematic parenchyma cells) and small cytoplasmic cells (primary meristematic cells). Large cells were very variable in size and shape. Most of them were rounded or ovoid and 80--100 pm in diameter. However, some were very elongated, sometimes spirally, and were several hundred pm in length. These large cells were generally aggregated into loose clumps that dissociated easily to give smaller aggregates and free cells. Small cells were generally isodiametric (about 20 ~m in diameter), densely cytoplasmic, with small vacuoles and large nuclei, and often filled with numerous starch grains. They formed compact clumps, which broke up during the suspension growth giving smaller groups that continued proliferating. These small cells seldom grew free. A third cell type, consisting of cells with plastids, was observed in green strains. Similar to the first cell type described, these cells had large vacuoles but were generally smaller (60--90 pm in length) and bowed in shape. They often developed as irregular files. As they aged, their plastids became filled with large starch grains. These 3 cell types were not always spatially separated and often constituted mixed clumps. Ontogenic relations between these various cells have not been studied. As previously stated by Maheshwari and Gupta (1965), cell suspensions behave like calli and produced embryoids when they were grown without 2,4-D (Fig. 1 a--e). Most of these embryoids developed from the periphery of clumps, where they formed clusters. They then became free-floating in the medium, being isolated or joined by their basal (root) end. Squashes of aggregates taken from recently induced embryogenic cultures showed young embryoids of various shapes and structures, with the first stages being linear or rounded, as in carrot (Halperin and Jensen, 1967; Backs-Hiisemann and Reinert, 1970; Halperin, 1970; Reinert et al., 1971; McWilliam et al., 1974; Smith and Street, 1974; Street and Withers, 1974). Their origin was not studied in detail. Culture o f plantlets obtained from embryoids In a first attempt, plantlets were transferred directly from their inductive medium (agar medium without 2,4-D) to a greenhouse soil. However, most of them withered, so that a multi-stage procedure had to be developed to improve their rooting and their resistance to wilting. To obtain mature plants from cell suspension we used the following process. Stage O: Cell suspension culture (see materials and methods). Stage 1 : Induction o f embryogenesis. -- At the end of Stage 0 (1 month), 2 ml of a stationary-phase culture was pipetted into 100 ml of an inductive medium and then shaken for 12--15 days in the same way as the suspensions. The new medium consisted of half-strength MS salts, 0.4 mg 1-~ thiamineHC1 and 1.5% glucose. During this stage, embryoids began their develop-
58
59
ment. The more advanced of them reached the cotyledonary stage and acquired individuality. This was also a diluting stage, later allowing better growth of plantlets on the Stage 2 medium. It was observed that the direct transfer of the Stage 0 cell suspension, even in a small quantity, on to a solid inductive medium often further impeded plantlet development because of the presence of too many embryoids. Embryoids did not give normal plantlets when maintained in this shaken inductive medium. Although they reached 2--3 cm in length, their stem remained atrophied and they did not resume normal development when transplanted on a solid medium. However, some of them did produce new adventive embryos which grew directly into plantlets. Stage 2: Development of plantlets from embryoids. -- After 12--15 days, one or two drops of the embryoid suspension were pipetted into 26 mm X 170 mm test tubes containing about 40 ml of a solid medium. The medium was almost the same as that of Stage 1, but contained 1% powdered charcoal (Fridborg and Eriksson, 1975; Drew, 1979) and 0.7% agar. The tubes were next closed with cotton plugs but not capped because capping caused yellowing of plantlets, delay in rooting and vitrification of some stems and petioles. The liquid medium film surrounding the embryoids disappeared a few days after the transfer, and they continued their development at the agar surface. This stage was approximately 10 weeks, the first plantle~s being transplantable after 4 weeks. Many of the plantlets formed by embryoids were completely teratologic and unusable. The number of these abnormal plantlets increased as the suspension aged. Generally, a 2-year-old culture was still embryogenic but produced no more normal plantlets. Only plantlets having normal leaves were transferred. Thus, the number of these transplantable plantlets varied with the strains and with their age. For some freshly initiated lines as many as 30 plantlets per tube were obtained. However, normally their number seldom exceeded 10. In our culture conditions, plantlets had a long hypocotyl and a thin primary root. As indicated previously (Hunault, 1981), these plantlets looked almost normal but they did show some differences from normal seedlings. (i) Cotyledons were generally shorter than normal, Fig. 1. Embryoid development during the induction stage (Stage 1, medium without 2,4-D). (a, b, c) Young embryoids torn away from their producing aggregate. In most cases, a few large elongated cells of the clump core strongly stick to the basal end of each embryoid. Some of these cells have large starch grains. Smaller grains are also seen in embryoid cells. (d) Cluster of large embryoids still attached to the producing clump. Embryoids are connected to the clump by large suspensor-like cells as in (a, b, c). (e) Free floating embryoid with t w o young well-developed cotyledons. (f) Shoot part of an embryoid showing only one cotyledon with a sheathing base. (g) Embryoid with two unequal cotyledons. (h) embryoid with two equal cotyledons (as in (e), but older). During further development, such cotyledons may elongate or remain short; s o m e of them become lobate.
60
sometimes reduced to a single piece (Fig. 1, f), unequal (Fig. 1, g) or with a divided lamina. (ii) Hypocotyls sometimes coalesced, sometimes up to the cotyledonary node. (iii) Leaf laminae of the first true leaves were less dissected than normal ones, and entire in some cases. E m b r y o and plantlet malformations have previously been described for embryogenic cultures of other umbelliferous species such as carrot (Halperin and Wetherell, 1964; Homes and Guillaume, 1967), parsley (Vasil and Hildebrandt, 1966) and caraway (Ammirato, 1974), and non-umbeUiferous species such as Citrus (Button et al., 1974). Such anomalies seem inherent to m a n y systems yielding vegetative embryos. Stage 3: Increase o f rooting and resistance to wilting. ~ Plantlets having at
least one true leaf (Fig. 2, a) were isolated and their hypocotyl cut about 1 cm under the cotyledonary node (Fig. 2, b). The long hypocotyl, the primary root and the collar, which were often distorted and showed a tendency to produce new adventive embryos during sub-culture, were thus discarded. Plantlets were then grown on a liquid medium made of halfstrength MS salts, 1.5% glucose and 0.1 mg 1-1 indolebutyric acid (IBA), the remnant of the hypocotyl being inserted through the central hole of a filter-paper dome dipping into the medium. New vigorous roots generally appeared on the hypocotyl 2--3 weeks later, while the stem gave new leaves (Fig. 2, c). Some plantlets bloomed (Fig. 2, d) and set several small fruit. However, these did not reach maturity and were sterile. Such rooted plantlets m a y be transferred to Stage 4 after 4--6 weeks. Not all plantlets were suitable for transplanting because some of them did not root, while the stem of others ceased growing and often became distorted. Between 60 and 70% could be transferred, although a higher percentage was obtained when rigorous selection was done at the end of Stage 2. Plantlet vigour is diminished when glucose concentration is higher than 2%. Badoc (1982) also stated that the best glucose concentration for bitter fennel micropropagation is about 2%. Increasing the IBA concentration to 0.5 mg 1-1 increased rooting and hardiness but caused distortion and callusing of the hypocotyl base. It also promoted the production of adventive embryos from the hypocotyl and cotyledons. As these modifications of the lower parts of plantlets were detrimental to further growth, the IBA concentration, although not optimal for rooting, was maintained at 0.1 mg 1-1
Fig. 2. Morphology of plantlets during sub-cultures. (a) Plantlets with a first true leaf isolated from Stage 2 cultures. (b) Identical plantlets prepared for Stage 3 culture (lower part discarded). (c) Rooted plantlet at the end of Stage 3. (d) Flowering plantlet at the end of Stage 3. (e) Young greenhouse grown plant after 2-month culture in Stage 4; a robust offshoot is developing from a lower node and will eventually substitute for the etiolated first stem. Such a substitution often occurs during Stages 4 or 5 and generally gives hardy plants.
2 i
1 •
|
3
I
4
|
5cm
62 TABLE I Influence o f soil mixture on plantlet establishment Number of pots
Substrate
Establishment rate (%) .
120 120 120
Vermiculite Vermiculite + peat (50:50) Perlite + peat (50:50)
83 66 36
S t a g e 4: G r e e n h o u s e t r a n s p l a n t a t i o n . -- Well-developed rooted plantlets were
removed from the tubes and transplanted into pots containing a synthetic soil wetted with a commercial mineral solution (Floreden). To avoid rotting during the first week, their lower part was sprayed with a 2% oxiquinoline solution (Cryptonol). Plants were further covered with a transparent polyethylene bag and transferred to a growth room (day period: 16 h, 15 000 lux, 22°C, 70% air saturation; night period: 8 h, 18°C, 95% air saturation). A drip system allowed each pot to be individually watered with the nutrient solution. It proved very important to avoid soil saturation, because the fennel root system is highly sensitive to asphyxiation. Plastic bags were removed after 5 days. Some plants withered, but about 70% kept growing and eventually flowered. The success of establishment and subsequent plant development varied with the soil mixture (Table I). Poor results were obtained with the perlite + peat substrate. The best establishment was obtained with vermiculite alone, but growth was better on vermiculite + peat (50:50). transfer. -- Plants were transferred to the field during spring or at the beginning of summer. Leaves which had been developed during Stage 4 often wilted after transfer, but after a period of adaptation, new robust shoots appeared on the lower part of the stems. They produced numerous umbellae during the following summer and some set seeds. No particular problem arose during this transfer, the success rate being generally higher than 80%.
S t a g e 5: O u t d o o r
DISCUSSION
The process described above allows the raising of mature bitter fennel plants from suspension cultures at a relatively high success rate. The field behaviour of more than 800 plants obtained in this way have been studied since 1980. Variations occurred in the plants, indicating that they could be of value in a breeding program. However, at the present time, three obstacles impede the progress of this work. The first obstacle is the progressive loss by suspensions of their ability to produce normal plantlets. The disappearance of the embryogenic potential of various carrot cultures has been recorded by
63 several authors (Reinert, 1959; Halperin and Wetherell, 1965; Mouras and Lutz, 1973; Smith and Street, 1974) and has been related to an increase in cell sensitivity to 2,4-D and to the competitive elimination of embryogenic cells by non-competent ones, these often being aneuploid or polyploid (Smith and Street, 1974). Culture of celery tissues in a shaken liquid medium also induced the loss of their embryogenic ability (A1-Abta and Collin, 1978). After several years, our fennel strains still produce embryoids but these develop later (most of them appear during Stage 2 instead of Stage 1) and all give completely abnormal plantlets. A detailed caryological study of suspensions has not yet been done, and it may be that it is caused by a chromosomal drift. However, this transformation is probably not related to a drift toward tetraploidy because recently, a tetraploid line was isolated (most cells tetraploid with 44 chromosomes or hypotetraploid) which was able to produce a high number of morphologically normal plantlets. Tetraploid carrots were obtained in this way by Smith and Street (1974). These authors stated that the decrease in embryogenic potential was frequently correlated with the dominance of cells having chromosome numbers above the tetraploid level. The second obstacle is that plants obtained from embryoids generally exhibit a very low level of fertility. Some of them prove completely sterile, even when cultivated near a field of normal plants, i.e. in an environment permitting good open pollination. Young fennel plants begin to produce umbellae during the greenhouse culture (sometimes in the test tubes at Stage 3) and flower profusely when field grown. After the shedding of perianth pieces and stamens, ovaries start growing larger, but most of them stop developing when they reach 5--6 mm in length; they then turn yellow and later abscise. Dissection shows that fruits become yellow only after ovule shrivelling. To our knowledge, such sterility has not been quoted for carrot, but fruits of celery plants raised from embryoids do not reach maturity (Merrick and Collin, 1980). Fennel and celery are closely related plants belonging to the same tribe and having the same chromosomic number (2n = 22), while carrot is an outlying species with 18 chromosomes. Hore observed various anomalies during male meiosis of fennel (1976} and celery (1977), and asserted (1979) that the poor seed-set of various aromatic umbelliferae is the result of bad pollen quality. He also stated that fennel may present some self-incompatibility (Hore, 1979). Although cultivars used at the present time in France generally yield a high number of seeds when field grown, our in vitro culture conditions could have induced cytogenetic irregularities causing plantlets to become either completely male sterile or selfincompatible. Nevertheless, this hypothesis cannot explain the poor yield of seeds when plants are open-pollinated. Aneusomatism could be another explanation. Ogura (1976), working on tobacco plantlets regenerated from callus, and Bennici and D'Amato (1978), on wheat plantlets obtained in vitro from mesocotyl, observed that these plantlets were mixoploid and had various numbers of aneuploid cells. Moreover, Browers and Orton (1982)
64
observed a significant number of hypodiploid cells in celery plantlets regenerated from embryoids. Caryologically heterogeneous plants of tobacco were generally less fertile than normal ones (Ogura, 1976). A preliminary study of the caryological status of several plantlets produced by our tetraploid line of fennel showed that they are also mixoploid, with various numbers of hypotetraploid cells. This aneusomatism could alter both male and female meiosis, thus leading to complete sterility. A detailed study of meiosis is needed to elucidate the reason for the almost total lack of fruiting of these plants raised from embryoids. The winter death of a high number of plants is the third obstacle impeding the progress of our work. In contrast to sweet fennel (Foeniculum vulgare Mill. ssp. capillaceum (Gilib.) Holmboe var. dulce (Miller) Thellung), bitter fennel is a perennial variety. Therefore, when beginning this study, we supposed that plants, once established during summer, could be studied for several years. Thus, it was disappointing to see in spring 1982 that about 90% of plants established during the 1981 season had died during the winter. This was observed in two experimental fields about 180 km apart. It is now of interest to study if this winter sensitivity is due to peculiar aspects of our cultural process or is ascribable to deeper modifications of the properties of these plants. ACKNOWLEDGEMENTS
I am greatly indebted to P. Desmarest, Institut de Recherches Appliqu~es aux Boissons, Cr~teil, France, for his invaluable help during the greenhouse and field culture of the fennel plants. I also wish to acknowledge A. Mahuet for reading the English text.
REFERENCES A1-Abta, S. and Collin, H.A., 1978. Control of embryoid development in tissue cultures of Celery. Ann. Bot., 42: 773--782. Ammirato, P.V., 1974. The effects of abscisic acid on the development of somatic embryos from cells of caraway (Carum carvi L.). Bot. Gaz., 135: 328--337. Backs-HUsemann, D. and Reinert, J., 1970. Embryobildung durch isolierte Einzelzellen aus Gewebekulturen yon Daucus carota. Protoplasma, 70: 49--60. Badoc, A., 1982. Contribution ~ l'~tude des ph~nom~nes de morphogen~se et de callogen~se de tissus de Fenouil vulgaire ( Foeniculum vulgate ssp. capiUaceum vat. vulgare (Mill.) Thellung), analyse des constituants de l'huile essentielle des explants. DEA, Universit~ des Sciences et Techniques, L~lle, 74 pp. Bennici, A. and D'Amato, F., 1978. In vitro regeneration of Durum wheat plants. I. Chromosome numbers of regenerated plantlets. Z. Pflanzenzuecht., 81: 305--311. Browers, M.A. and Orton, T.J., 1982. Transmission of gross chromosomal variability from suspension cultures into regenerated celery plants. J. Hered., 73: 159--162. Button, J., Kochba, J. and Bornman, C.H., 1974. Fine structure of and embryoid development from embryogenic ovular callus of 'Shamouti' orange (Citrus sinensis Osb.). J. Exp. Bot., 25: 446--457.
65 Desmarest, P., 1978. N e w aspects of fennel cultivation in France. Acta Hortic., 73: 289-295. Drew, R.L.K., 1979. Effect of activated charcoal on embryogenesis and regeneration of plantlets from suspension cultures of Carrot (Daucus carota L.). Ann. Bot., 44: 387-389. Fridborg, G. and Eriksson, T., 1975. Effects of activated charcoal on growth and morphogenesis in cell cultures. Physiol. Plant., 34: 306--308. Halperin, W., 1970. Embryos from somatic plant cells. Symp. Int. Soc. Cell Biol., 9" 169--191. Halperin, W. and Jensen, W.A., 1967. Ultrastructuralchanges during growth and embryogenesis in carrot cellcultures. J. Ultrastruct. Res., 18: 428--443. Halperin, W. and Wetherell, D.F., 1964. Adventive embryony in tissue cultures of the wild carrot, Daucus carota. A m . J. Bot., 51: 274--283. Halperin, W. and Wetherell, D.F., 1965. Ontogeny of adventive embryos of wild carrot. Science, 147: 756--758. Homes, J.L.A. and Guillaume, M., 1967. Ph~nom~nes d'organogen~se dans des cultures in vitro de tissusde carotte (Daucus carota L.). Bull. Soc. R. Bot. Belg., 100: 239--258. Hore, A., 1976. Cytogenetical studies of the genus Foeniculum (Umbelliferae). Indian Agric., 20 : 183--191. Hore, A., 1977. Study of the structure and behaviour of chromosomes of the different varietiesof Apium graveolens (Celery). Cytologia, 42: 21--28. Hore, A., 1979. Improvement of minor (Umbelliferous) spices in India. Econ. Bot., 33: 290--297. Hunault, G., 1981. La culture in vitro des tissus de Fenouil (Foeniculum vulgate Miller). Premieres observations sur le c o m p o r t e m e n t des explantats primitifs et des cals. C.R. Acad. Sci. Paris, Set. III, 293: 553--558. Linsmaier, E.M. and Skoog, F., 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant., 18: 100--127. Maheshwari, S.C. and Gupta, G.R.P., 1965. Production of adventitious embryoids in vitro from stem callus of Foeniculum vulgare. Planta, 67 : 384--386. McWilliam, A.A., Smith, S.M. and Street, H.E., 1974. The origin and development of embryoids in suspension cultures of carrot (Daucus carota). Ann. Bot., 38: 243--250. Merrick, M. and Collin, H.A., 1980. Selection for resistance to the herbicide Asulam using plant tissue cultures. In: Cong. Eucarpia, Application de la culture in vitro ~ l'am~lioration des plantes potag~res, 16--18 April 1980, Versailles, France, pp. 121--125. Mouras, A. and Lutz, A., 1973. Sur les variations du comportement organog~ne des cultures de tissus de Carotte sauvage produisant des pseudo-embryons. C.R. Acad. Sci. Paris, S~r. D, 177: 1151--1153. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473--497. Ogura, H., 1976. The cytological chimeras in original regenerates from tobacco tissue cultures and in their offspring. Jpn J. Genet., 51: 161--174. Reinert, J., 1959. Uber die Kontrolle der Morphogenese und die induktion yon Adventivembryonen an Gewebekulturen aus Karotten. Planta, 53 : 318--333. Reinert, J., Backs-HUsemann, D. and Zerban, H., 1971. Determination of embryo and root formation in tissue cultures from Daucus carota. In: Coll. Int. CNRS No. 193, Les cultures de tissus de plantes, 6--10 July 1970, Strasbourg, France, pp. 261--268. Smith, S.M. and Street, H.E., 1974. The decline of embryogenic potential as callus and suspension cultures of Carrot (Daucus carota L.) are serially subcultured. Ann. Bot., 38: 223--241. Street, H.E. and Withers, L.A., 1974. The anatomy of embryogenesis in culture. In: H.Ep Street (Editor), Tissue Culture and Plant Science 1974. Academic Press, London, pp. 71--100. Vasil, I.K. and Hildebrandt, A.C., 1966. Variations of morphogenetic behavior in plant tissue cultures. II. Petroselinum hortense. Am. J. Bot., 53: 869--874.