9

IN VITRO STRATEGIES FOR TROPICAL FRUIT TREE IMPROVEMENT

Richard E. Litz

ABSTRACT

Vegetatively propagated tropical fruit trees can be highly sensitive to plant disease epidemics. These trees are usually very heterozygous and have long generation cycles (e.g., 7-20 years). Some tropical fruit species such as mangosteen, which is obligately apomictic (nucellar embryony), are recalcitrant to conventional plant breeding approaches. Strategies for the recovery of horticulturally useful plants from cell and tissue cultures have great potential for the improvement of tropical fruit species. De /lOVO regeneration pathways have been described from explants of selected mature trees of perennial tropical fruit species in Anacardiaceae, Euphorbiaceac, Moraceae, Myrtaceae, Oxalidaceae, Rosaceae, Rubiaceae, Rutaceac, Sapindaceae and

Richard E. Lit::: * Tropical Research and Education Center, University of Florida, 18905 S.W. 280 St.. Homestead. Florida. 33031 U.S.A.

f'lorida Agricultural Experimental Station Journal Series No. 8677.

V. Dhawan (ed.), Applications of Biotechnology in Forestry and Horticulture© Plenum Press, New York 1989

110 Litz

Sterculiaceae. The regeneration of tropical fruit trees and the application of the approaches for cultivar improvement are the subjects of this discussion.

INTRODUCTION

Perennial tropical fruit species are important in the Third World as additional sources of revenue through export and as important diet supplements. However, there has been scarcely any conscious effort directed towards cultivar improvement through classical plant breeding. Consequently, outstanding fruit trees that have occurred by chance have been vegetatively propagated and maintained in orchards of the same genotype. In the ancient civilisations of south and south-east Asia, some of these tree selections are several hundred or more than one thousand years old. Vegetative propa­gation of these selections has preserved the unique genetic composition that confers outstanding horticultural characteristics.

The limitations of monoculture in tropical agriculture have become painfully evident during the past century, with successive disease epidemics affecting the pro­duction of banana and plantain (Mllsa), cacao (Theobroma cacao), coffee (Coffea ar­abica), papaya (Carica papaya) and coconut (Cocos ll11cifera) in the neotropics. In response to these challenges, the genetic improvement of tropical fruit trees by con­ventional plant breeding approaches has been confounded by the long generation cycle of these plants, the heterogeneity of superior tree selections, the absence of continuing breeding and genetic studies and the inaccessibility or inadequacy of germ plasm re­positories.

There are certain common cultivar improvement or breeding objectives that can be designated for many tropical fruit tree species. Among these, disease and pest resistance have traditionally held high priority. The control of virus diseases of Citnts has been accomplished by the use of micro-grafting. (Navarro, 1981); however, the control of fungal and bacterial pathogens has continued to be dependent on chemical applications or more drastic measures such as quarantine and plant destruction, in order to prevent the spread of threatening pathogenic micro-organisms. The occur­rence and spread of crop-threatening diseases being extremely rapid, conventional breeding strategies are inadequate to meet the challenge of these epidemics involving tropical fruit trees. Consequently, highly esteemed tropical fruit cuItivars that lack the necessary genetic protection against pathogens are usually removed from cultivation and are replaced by horticulturally inferior selections.

Other breeding goals must include the development of improved rootstocks that can tolerate soil-borne fungi, saline conditions and/or those that will confer a dwarf habit to tropical fruit trees, many of which originated in the tropical rain forests as tall canopy trees. Many other breeding imperatives remain, e.g., overcoming alter­nate bearing, improving fruit quality, extending the geographical range by selection for cold tolerance, etc. However, these objectives cannot be achieved readily by using either traditional approaches or currently available biotechnology strategies. It is

9. Tropical Fillit Tree Improvement 111

anticipated that procedures for producing transgenic plants will be greatly improved in the next few years, together with identification and cloning of horticulturally useful genes.

The development and application of ill vitro strategies for tropical fruit tree cultivar improvement has been reviewed previously by Litz et al. (1985) and by Litz (1985; 1987). The potential use of cell and tissue culture techniques as integral components in tropical fruit tree breeding certai.nly has great appeal. The ability to manipulate the genome of well-established fruit tree cultivars, and to direct the breeding effort toward specific goals such as disease or pest resistance, would obviate the sexual process and the long (7-20 years) juvenile cycles that would ensue. The basic nature of the cultivar could be maintained, although certain traits could be altered in a discrete manner. The use of ill vitro approaches for tropical fruit cultivar improvement is, however, predicated on the assumption that regeneration from somatic tissues of the mature tree is possible.

PERMISSIVE PATTERNS OF REGENERATION

Regeneration from callus derived from somatic tissues of mature tropical fruit trees has, in fact, been the most limiting factor in the application of biotechnolob'Y to these species. The morphogenetic potential of explants from juvenile or emhryonic tissues of angiosperm trees has been well-characterised. However, until recently, rela­tively few tree species have been regenerated from cell or callus cultures derived from mature trees. Ironically, somatic embryogenesis from cultured ovules of polyembryonic Citnts sp. was described some thirty years ago by Stevenson (1956). In subsequent studies by Maheshwari and Rangaswamy (1958), Rangaswamy, (1961) and Sabharwal (1962), this regeneration pathway was described in greater detail. Somatic embryogenesis or embryogenic pseudobulbil callus appeared to have developed di­rectly from adventitious embryos already present in the nucellar explant. This pattern of direct somatic embryogenesis from nucellar explants was confirmed by Rangan et al. (1968) with monoembryonic Citnts sp. Button et al. (1974) showed that the Citl1lS pseudobulbil callus was entirely composed of small globular proembryos at various stages of development. Hence, a true unorganised embryogenic Cilnts callus may not occur, and even if it does, it will have a brief existence. Other studies have indicated that, unlike somatic embryogenesis in DallClls carota, the process involving Cilms nucellar tissue is not dependent on the presence of an auxin in the medium (Murashige and Tucker, 1969; Kochba and Spiegel-Roy, 1977; Tisserat and Murashige, 1977). Although the regeneration of Citnts by somatic embryogenesis from nucellar tissues has been well-characterised for many years, it did not serve as a model for other tropi­cal trees until recently. Tropical tree species have been regarded to be somewhat recalcitrant ill vitro. Using approaches adapted from previously reported CilntS ill vitro studies, several tropical fruit tree species representing several plant families have been regenerated from tissue cultures derived from cultured nucelli (Table 1). Somatic

112 Litz

Table 1: Permissive somatic embryogenesis from the nucellus of tropical fruit trees

Species Common name Family Reference

CitlHs sp. citrus Rutaceae Stevenson ( 1956)

Elio/wl!ya japon ica loquat Rosaceae Litz (19R5)

Eugenia sp. rose and malay apples Myrtaceae Litz ( 1984b)

Mangifera indica mango Anacardi- Litz et a!. aceae (1982)

Myrcimia caulij70ra jaboticaba Myrtaceae Litz (1904c)

embryogenesis in all of these species is not dependent on exogenous growth regulators and occurs directly from the nueellar explant or by secondary budding from globular proembryos.

Nucellar Cells of many species evidently possess the ability to produce somatic embryos without the influence of an external stimulus. This regeneration pathway has been described to involve pre-embryogenic determined (PED) cells (Sharp et aI., 1980). The PED cells only require release from the inhibitory environment of the ovule to become embryogenic. Ammirato (1987) has defined somatic embryogenesis from PED cells as being permissive, because the nucellar cells are already fully competent to produce somatic embryos.

Is the nucellus the explant of choice for regenerating tropical fruit trees? Per­missive somatic embryogenesis from the nucellus of tropical trees is generally re­stricted to species with relatively large seeds, genera in which the incidence of nucellar polyembryony is notable and species within a narrow range of plant families. It is prob­able that many tropical fruit tree species could be regenerated by this pathway. Ulti­mately, this must be determined for each species and cannot be predicted.

Cultivar or genotype can influence somatic embryogenesis from the nucellus. This has been observed not only in citrus (Moore, 1986), but also in mango (Litz, 1984a; 1987). There are significant differences in the responses among cultivars and between polyembryonic and monoembryonic cultivars of the same species. Even within a cultivar, there are different morphogenetic responses from cultured nucelli that are dependent on the stage of development of the ovule at the time of explanting (Fig. 1). In mango, the optimum stage of development of the ovule for establishing embryogenic cultures corresponds to that period during which the embryo mass occu­pies approximately one half of the embryo sac. This period also corresponds with high endogenous concentrations of the polyamines (putrescine and spermidine) in the nucellus (Litz, 1987). The interaction between the growth medium and the nucellar explant is also very important. Although somatic embryogenesis can occur directly from the excised nucellus on medium without growth regulators, the presence of 2,4-0 can stimulate the process of regeneration, presumably by cloning the PED cells (Litz, 1987).

9. Tropical FHlit Tree Improvement 113

40

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O+-----~----~----_r----_+----_+----~----__. o 1 2 3 4 5 6 7

Embryo Category

FiJ.:. I: Effect of ovule development on somatic embryogenesis from nllcellar explants of mango. Embryo categories refer to the ratio of embryo length: ovule length. Category I-ratio 0.15; Category 2-0.2; Category 3-0.4; Category 4-0.6; Category 5-0.8; Category 6: 1.0.

INDUCTIVE PATTERNS OF REGENERATION

The de IlOVO regeneration of tropical fruit tree species from tissues other than the nucellus has only recently been described (Table 2). Young leaves in fresh vegeta­tive flushes of certain fruit species can be induced to produce an unorganised regen­erative callus on growth media containing 2,4-0 and a cytokinin.

The induction of embryogenic callus in EliphOlia lOllgall is strongly cytokinin­dependent; regeneration can only occur if kinetin is present in the induction medium. Similar conditions promote the formation of embryogenic callus in the tropical forest trees Sapilldlls tri/oliatlls (Desai et aI., 1986) and in CoJJea arabica (Son dahl and Sharp, 1977). Interestingly, the media that induce morphogenesis in leaf callus of E. lOllgall and S. tli/oliatlls have no such effect on leaf explants of the closely related Litchi chillellsis (Litz, unpublished data). Genotype, in addition to medium, consequently has an important role in realising the morphogenetic potential of cultured leaves of tree species in the Sapindaceae.

114 Litz

Table 2 : Inductive Morphogenesis from somatic tissues of mature tropical fruit trees

Species Common Family Regeneration Reference name pathway

AvenilOa carambola carambola Oxalidaceae organogenesis Litz & Griffis (1988)

EuphO/ia tOllgall longan Sapindaceae somatic Litz (1988) embryogenesis

MOllls illdica mulberry Moraceae organogenesis Mhatre et al. (1985)

Solallum quitoellse lulo Solanaceae organogenesis Hendrix et al. (1987)

Regeneration from leaf callus of E. IOllgall is consistent with the description of inductive somatic embryogenesis (Ammirato, 1987) because a change in competency of leaf cells has occurred in response to the stimulus of an auxin and a cytokinin. This regeneration pathway is distinct from that described for nucellar explants. Implicit in this response is the induction of a subculturable, unorganised callus.

De 1I0VO regeneration via organogenesis has also been described from callus de­rived from young leaves ofAverrllOa carambola (Litz and Griffis, 1988), Solanum qui­toellse (Hendrix et al., 1987) and Monts indica (Mhatre et al., 1985). This regeneration pathway is also an example of inductive morphogenesis, as leaf cells undergo a change of competency to form adventitious meristems in the presence of an auxin and a cy­tokinin.

APPLICATION OF IN VITRO SYSTEMS

With the exception of Citnts spp., there have been relatively few attempts to utilise in vitro systems to address important cuItivar improvement problems. This is largely due to the fact that regeneration protocols have been described only recently. However, there are also considerable problems associated with stimulating of normal maturation and germination of somatic embryos of tropical fruit tree species having large seeds. Mature somatic embryos of mango can attain length of 4.0 cm at the time of germination. In vitro conditions for controlling maturation and germination of somatic embryos in mango (DeWald, 1987) and longan (Litz, 1988) have recently been described. It is necessary to prevent precocious germination of these somatic embryos by altering the physical conditions required for growth and by manipulating the growth medium during maturation (DeWald, 1987; Litz, 1988; Fig. 2).

Research involving Citnts has attempted to address certain breeding problems using two approaches:

* ill vitro selection of somaclonal variants for salt tolerance, 2,4-0 resistance and resis­tance to mal secco toxin; and

* protoplast fusion between Citnts sp. and related genera in order to produce disease resistant, frost resistant, dwarfing rootstocks.

9. TlVpical Fltlit Tree ImplVl'emelll 115

30

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20 E CD

0 6. "0 15 E 6.

• • • 6.

6. • 0

'" .... 10 0 ...

• .. CD .0 E 5 ::J

• • • Z

0 0 2 3 4 5 6 7

Length of somatic embryos (cm)

Fig. 2: Relationship between precocious maturation of mango somatic embryos and abnormal development. Somatic cmbryos with roots and shoots; somatic embryos with roots only. (Sample size = 202 somatic cmbryos).

Exploiting the genetic instability that is known to exist in cell and tissue cultures, i.e., somaclonal variation, Kochba et ai. (1980; 1982) were able to recover embryogenic cell lines of 'Shamouti' and sour orange calli that were apparently resistant to sodium chloride concentrations up to 10.0 gl-l. None of the resistant lines, however, could be stimulated to produce embryos. A similar approach was adopted to select for height­ened resistance to the herbicide 2,4-0 in callus of 'Shamouti' orange (Kochba et ai., 1980; Speigel-Roy et ai., 1983). Callus with resistance to 10M 2,4-0 was selected; however, plants were not regenerated.

The recovery of disease resistant tropical fruit trees derived from somaclonal variants in the selection medium has been a high priority. Efforts have been made to select for specific disease resistant Cit11ls by exposing embryogenic callus to the toxin associated with 11Ial secco disease (Nachmias et ai., 1977). Another approach has been adopted for mango. Allthracllose, a fruit and foliage disease of mango, is caused by the fungus Colletotricl1ll11l gloeospOIiodes. The disease is particularly severe in the humid tropics. The monoembryonic Indian and Florida cuhivars are generally highly susceptible to allthracllose, whereas the polyembryonic south-cast Asian cuhivars have considerable resistance to this pathogen. It is evident that resistance to allthracllose is present within the species, but transfer of resistance to monoembryonic mango cultivars with superior horticultural quality has not been undertaken. The infection of plant tissues by Colletotricl1ll11l sp. is accompanied by the release of a polysaccharide elicitor from the fungal walls, which triggers a hypersensitive response within the host tissue (Anderson-Prouty and Albersheim, 1975). The hypersensitive response is characterised by the accumulation of phytoalcxins in the tissue and is always

116 Litz

accompanied by the browning of the tissue. Partially purified extracts derived from culture filtrates of Colletotricl1ll11l sp. will also elicit the same response. It has recently been demonstrated that the fungal culture filtrate can be incorporated into the tissue culture medium, in which it can trigger a similar hypersensitive response as reported in cell cultures of the forage legume Stylosanthes sp. (Lopez et aI., 1987). Plants regenerated from the cells that survived this selection pressure were found to be more resistant to anthracnose (Lopez et aI., 1987). Preliminary studies in which mango callus was exposed to the partially purified culture filtrate of Colletotrichll11l gloeosporiodes have demonstrated that a hypersensitive response is elicited within 48-72 hours (Litz, unpublished). It is hoped that this approach can be utilised for cultivar improvement among the monoembryonic mangos.

The application of protoplast technology to tropical fruit trees has only been re­ported for Cit11ls sp. Vardi et al. (1975) first reported the successful recovery of so­matic embryos from protoplasts derived from Cit11lS nucellar callus. In subsequent studies, the conditions for improving Cit11ls protoplast plating efficiency were greatly improved and the range of cultivar responses was measured (Vardi, 1981; Vardi et aI., 1982). Inter-generic somatic hybridisation between Cit11ls sinensis and Ponci11ls trifoliata (Ohgawara et aI., 1985) has been achieved and the parasexual hybrid plants have been regenerated. Using similar protocols, Grosser et al. (1988a,b) have created artificial hybrids between several Cit11ls spp. and species of other related genera, thereby anticipating the development of suitable germplasm for use as dwarfing root­stocks.

CONCLUSION

Considerable progress has been made in overcoming the problems associated with morphogenesis in callus derived from explants from mature tropical fruit trees. This should facilitate the application of modern genetics to the improvement of this important group of plants.

ACKNOWLDEGEMENTS

The author acknowledges with gratitude the assistance of Ms Roe C. Hendrix and Callie Sullivan.

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9. Tropical Fmit Tree Improl'ement 117

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