HortResearch Publication - Developing New Seedless Citrus Triploid Cultivars
Excessive seed numbers in citrus fruit are a real consumer turn-off. In our search for better easy peel hybrids for export and local markets, we have begun using plant tissue culture techniques to develop seedless triploid selections.
In 1983, the New Zealand Citrus Industry Planning Council developed a set of citrus breeding goals tailored specifically to the needs of the New Zealand citrus industry. These goals were specifically to create new easy peel hybrids which produced export quality fruit under New Zealand orchard conditions.
A conventional breeding programme is now well under way, but as market requirements are redefined, we need to incorporate new aims into this programme. One specific aim we are now pursuing is seedlessness.
Seedy citrus fruits are fast becoming unacceptable on international markets, and our taste panel experiences with local consumers show that they also find citrus seeds unpleasant. It is becoming increasingly important that any new easy peeler hybrids we develop have a high degree of seedlessness.
We have also needed to take into account the mixed orcharding approach that is used locally, where many citrus cultivars are often grown in close proximity. Some citrus cultivars will be almost seedless if they are planted in large solid blocks, because they are self sterile (Clementine mandarin and NZ grapefruit are two examples). They will unfortunately become seedy if pollinated by another cultivar. We have chosen to use in our breeding programme a strategy for seedlessness called triploidy that will work even where cross pollination occurs.
Polyploidy is the condition in which each cell contains three or more sets of chromosomes. This occurs naturally in citrus, often through spontaneous mutations.
Changes to the ploidy level of citrus can affect the size, habit and vigour of the tree and the quality and seediness of the fruit. For example, a tetraploid (4n) citrus plant has 4 sets of chromosomes and typically has stouter stems, thicker more leathery leaves and is less vigorous than its diploid counterpart (Gmitter and Ling, 1991). Examples of tetraploid citrus are often found in a batch of citrus rootstock seedlings.
Figure 1: Seedless fruit of the Tahiti lime
Although the reduced vigour of citrus tetraploids does limit their development as a crop, they are invaluable in breeding programmes which are aiming to develop new dwarfing rootstocks, or for backcrossing with diploid species to produce triploid plants (3n).
Unfortunately the frequency of naturally-occurring triploids in Citrus is low. It is possible, however, to produce triploid citrus plants either by regeneration in tissue culture of triploid plantlets derived from triploid endosperm tissue, or by backcrossing a tetraploid (4n) with a diploid (2n) to generate triploid offspring (Figure 2).
The latter technique has been used by the University of California in breeding two seedless pummelo x grapefruit hybrids, Oroblanco and Melogold (Soost and Cameron; 1980, 1985).
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| Figure 2: The development of triploids by interploid hybridisation | Figure 3: Stages in embryo development (not to scale) |
These techniques and the inherent seedlessness of triploid citrus types offers the possibility for the induction of seedlessness in future breeding lines and a number of 'seedy' but otherwise excellent commercial cultivars.
In our citrus breeding programme we aim to produce triploid plants in selected cultivars using plant tissue culture techniques. Two approaches have been taken:
Somatic embryogenesis is the process where embryos are formed in tissue culture and there are two distinct steps. The first, the induction of embryonic cells or proembryoids, is followed by the development of these proembryoid cells into embryoids. The embryoids follow a developmental sequence similar to that of an embryo in a seed. The sequential stages are from small cell aggregates to globular, heart-shaped and torpedo-shaped embryoids to complete plantlets (Figure 3).
Therefore the use of triploid endosperm tissue could prove a quick and efficient method of obtaining triploid citrus plants. The drawback to using this method is that the window for obtaining endosperm tissue from immature citrus seeds is limited to only three weeks in each year, and success rates have been very low. Three cultivars were used in this study; Dweet tangor, Encore mandarin and Umatilla tangor.
All stages of embryo development were observed and complete plantlets were obtained. Several of these plantlets had an abnormal morphology and there was a high level of organ fasciation where the shoots appeared to be fused. This type of aberration is commonly associated with triploid plants.
Unfortunately these treatments are commonly associated with chimeras, ie tissue containing mixed populations of cells. In colchicine treatments the chimeras consist of cells of different ploidy levels but in other chimeras the cells may vary in their pigmentation. In the latter case this may be visible as a band on the fruit rind which is lighter or darker than the surrounding rind. Chimeras are usually unstable and during development the cells revert back to the natural or original condition.
By using tissue culture methods the incidence of chimeras is reduced compared to treatments where the colchicine is applied to young shoots. This is because the plantlets regenerated using tissue culture are derived from a few cells compared to the many-celled shoots.
Once we have obtained tetraploids it is then necessary to use these plants in further cross-fertilisation experiments in order to produce triploids. Therefore obtaining triploids by this method is more labour intensive and time consuming than the regeneration of triploid plants from endosperm.
We have successfully regenerated a large number of plantlets via somatic embryogenesis from endosperm tissue of Dweet tangor. The ploidy level of all plantlets will be assessed by cytogenetic techniques and confirmed triploid or tetraploid plantlets will undergo shoot-tip grafting onto vigorous rootstocks.
Experiments will be continued with modifications to the growth media and the time period during which the fruitlets are collected. In future, we will carry out controlled pollinations between parents with desirable traits and culture the endosperm from the resulting fruitlets.
Gmitter, F.G., Jr., Ling, X. (1991) Embryogenesis in vitro and nonchimeric tetraploid plant recovery from undeveloped Citrus ovules treated with colchicine. J. Amer. Soc. Hort. Sci 116: 317-321
Soost, R.K., Cameron, J.W. (1980) 'Oroblanco' a triploid Pummelo-grapefruit hybrid. HortScience 15 :667-669
Soost, R.K., Cameron, J.W. (1985) 'Melogold' a triploid Pummelo-grapefruit hybrid. HortScience 20 :1134-1135
Source: The Orchardist, February 1995