Andrasfalvy, Andras

      Experimental Allogamy of the Tomato “EXALTO” - a new multi-purpose breeding procedure.

MATING SYSTEM

      Tomato belongs to the largest group of cultivated plants being predominantly self-fertilizing. The rate of

allogamy does not exceed generally 1 per cent. There is a developed self-pollination mechanism which is

claimed to be important for stable yields under a wide range of climatic conditions.

      The stigma, which is well protected in a tight column of anthers, is already receptive when pollen has

been shed. In the normal, pendent position of the flower, pollen flows downward and is prevented from

escaping by the rim of the stigma. Thus self pollination is achieved with maximal safety.

      In Central Europe (e.g. Hungary) the only insects active in visiting flowers of tomato are bumble bees

(Bombus spp.), which collect pollen by hanging on the flower, buzzing by the wing muscles and biting vigorously the anther tube in order to stimulate pollen release. Pollen accumulates on the hairy ventral surface of

the insect and is brushed into the basket of the hinder leg. When the next flower is visited, there is a low

chance of pollen carried along reaching the stigma and effecting allogamy. Accessibility of the stigma is,

however, crucial. Extruding stigma as a heritable character impairs the self pollinating mechanism, reduces

fertility, thus it is undesired in production.

ENFORCED ALLOGAMY

      What is the purpose of allogamy in a species with an established self-fertilization mechanism? As a

matter of fact, self fertilization has many more advantages than allogamy even at lower frequencies: safe fruit

set, uniform population true breeding, distinct cultivar characters. Whereas breeders are obliged to observe the pedigree or backcross methods with rather restricted possibilities of recombination, but they enjoy the bonuses of easy isolation and multiplication procedures.

      There is, however, a very popular modern method of breeding which needs total cross fertilization in masses of commercial seed scale: production of F₁ HYBRIDS. Tomato is claimed to be an ‘easy’ plant for

hand pollination because of high frequency of fruit set and high number of seed per fruit. With the growing popularity of F₁ hybrids, however, traditional procedures of hybrid seed production became a limiting factor and the improvement of efficiency is urgently necessary.

MALE STERILE (MS)mutants have been suggested repeatedly in hybrid seed production, but a slight reduction of fertility in F₁ prohibited their extensive use. Moreover, insects appear to reject male sterile flowers, because there is no pollen available, therefore they avoid ms plants after some vain attempt of foraging.

      FUNCTIONAL STERILITY (FS) seems to be a promising alternative to produce synchronous mass allogamy in tomato. FS plants produce normal gametes, but the self-pollination mechanism is modified, thus self fertilization is more or less suppressed or impaired up to complete (functional) sterility, whereas foreign pollen is allowed to reach the stigma in a less closed (dialysed) anther tube.

      Two recessive genes are responsible to perform the desired modification: ps-2 (positional sterile) preventing dehiscence of the anthers and dl (dialytic) suppressing development of hairs connecting the flanks of anthers to an anther tube. FS plants must be equipped, moreover, with recessive MARKER GENES facilitating the recognition of outcrosses in an early seedling stage, provided that potential pollen sources represent the “wild type”, i.e. carry dominant alleles of the marker genes.

      MULTIPLICATION of FS plants requires forced self pollination substituting the modified self pollinating mechanism. Insect visits may contribute to self pollination as well, because bumble bees bite the anther tube. Thus, under insect proof conditions no fruit set is expected, unless artificial pollination ensues.

      HYBRID SEED production with collected pollen promises high efficiency, though possibilities of the economic exploitation of insect mediated pollination seems to be still promising.

      As expression of the FS character is modified by environment as well as by the genetic background, in other words polygenes, further SELECTION of FS lines as well as their transfer to potential maternal parents of current F₁ hybrids and cultivars is required. That’s because experimental monitoring of the incidence of ALLOGAMY is continuously necessary. The present design is developed for this purpose.

EXPERIMENTAL DESIGN

      The PRINCIPLE: FS plants are planted in alternate hills with normal, i.e. “wild type” plants, as potential sources of pollen in order to offer opportunities of cross pollination performed by insects. Fruit set is expected to be from less abundant than normal to very scarce, depending on insect (bumble bee) population as well as on synchronous anthesis of FS and normal phenotypes. The incidence of wild phenotypes in the progenies produced on the FS plants is a measure of allogamy and/or of the efficacy of functional sterility. Ratio

of outcrosses is easily determined by telling marked plantlets (germs or seeds) from unmarked ones in trays or in petri dishes.

      RAISING TRANSPLANTS: FB lines have to be seeded in trays or seed beds of an easy access. About 10 days after emergence wild type seedlings have to be discarded as undesired outcrosses (indicating the allogamy of the former generation), saving “selfs” for planting. Keeping plantlets relatively dry and cool ensures clear distinction in expanded cotyledon stage, i.e. bright purple (wild) versus green (marked) color, normal (wild) versus dwarf (marked) length of the hypocotyl.

      PLANTING TO THE FIELD should be timed in the spring according to local practices (a final diagnosis of the marked FS plants when buds display the dialytic anthers is desirable).

      The PLANTING DESIGN should either follow the local practice or the intra row distance may be increased in order to distinguish easier neighboring plants. Staking is preferred if possible.

      REPLICATIONS and BORDER PLANTING of the pollinator is preferred to complete the field plots, where individual FS plants will furnish data of intra-plot variation. Orientation to hedgerows or other competitiveplantings should be taken in to account.

      It is desirable to repeat the experiment at several SITES and SEASONS. The specific method must be determined by local conditions.

      FS and pollinator plants should be planted alternately on neighboring hills in order to prevent insects from traveling in straight lines of only normal plants.

      For SYNCHRONOUS ANTHERS of FS and pollinator plants the latter should be either continuously growing (indeterminate) cultivars or their inflorescences should be repeatedly pruned in order to prevent fruit set and to keep plants growing throughout the summer season. Most of the available FS lines are determinate (self pruning), but the delay of fruit set stimulates branching and prolonged flower production. Bumble bee population is building up and/or concurrent pollen sources of wild flowers are reduced during July, when current determinate cultivars usually stop flowering.

      HARVEST of ripe or green but developed fruits should be made from each FS plant separately. Copious fruit set of the first 3 or 4 inflorescences indicates rather normal fertility, thus those plants should be discarded. Sterile first inflorescences followed by almost normal set of fruit during July may be a safe indication of bumble bee activity on FS plants.

      FRACTIONATE HARVEST may facilitate experimental approach of seasonal effects on allogamy within the same plant.

      MARKED (anthocyaninles5) plants of normal fertility are undesirable since they threaten the success of

the allogamy test, therefore different, non allelic markers (a, ah, aa, aw ) should be varied in neighboring rows of Planting, thus complementary action results in wild type hybrids between non allelic homozygotes. Alternative or additional markers are dwarf(d), lutescent(l) and brown seed (bs, bs-2).

      Evaluation of the experiment could be begun during the winter season in the laboratory by counting brown seed and/or germinating seed samples on moist filter paper. Just after the appearance of the radicle

natural daylight and low (15 to 20°C) temperature stimulates anthocyanin formation of the ‘wild” phenotype. For comparison normal cultivar (pollinator) seed should be represented amongst the samples. The same result is expected from dense seeding in trays, pots or nursery beds, though temperature and light regulation as well as close inspection may become more circumstantial Dwarf and lutescent phenotypes however can not be identified in petri dishes efficiently. Lutescence must be “developed” by stress, preferably by drought.

      Marked “selfs” of the best allogamous plants, with the highest incidence of wild type progeny, Should be saved for the next years experiment. Reliable lines should be MULTIPLIED by hand pollination using their own pollen.

      The TRANSFER of FS into new genotypes by backcrossing has to be calculated with the consideration of at least three independent genes (ps-2, dl and marker), i.e. the ratio of the desired recombinants in F2 is expected to be less than 1/64.

      Planting design for example:

S = Functional sterile marked 1

Z = Functional sterile marked 2

X = Functional sterile marked 3

Y = Functional sterile marked 4

P = Pollinator variety

      P     PP    PP    PP    PP    PP    PP    etc=margin plants

      P     SP    ZP    XP    YP    SP    ZP    etc

      P     PS    PZ    PX    PY    PS    PZ    etc

      P     SP    ZP    XP    YP    SP    ZP    etc

      P     PS    PZ    PX    PY    PS    PZ    etc

      etc.                                     

REMARK:     Regarding the considerable labor needed, restricted number of replicates and FS plants perplots are suggested. Maximal care should be given to the selection of FS plants, i.e. once the seedling marker is state, anther tube should be checked as for being dialytic. Backcrosses to wild type half sibs

should be absolutely avoided.

For further details: A. Andrasfalvy,gudapest Tigris U. 33., Hungary. H-1016