Watermelon Research Articles

Seedless Watermelon Breeding

Fred McCuistion and Todd C. Wehner

Unpublished

Tetraploid Production

Use of triploid hybrids has provided a method for production of seedless fruit. Kihara began working on seedless watermelons in 1939, and had commercial triploid hybrids available 12 years later. The development of triploid cultivars adds several problems to the process of watermelon breeding: extra time for the development of tetraploids; additional selection against sterility and fruit abnormalities; choice of parents for reduced seed coat production; the reduction in seed yield per acre obtained by seed companies; reduced seed vigor for the grower; and the necessity for diploid pollenizer taking one-third of the grower's production field.

Seedless cultivars are produced by crossing a tetraploid (4X=44) inbred line as the female parent with a diploid (2X=22) inbred line as the male parent of the hybrid. The reciprocal cross (diploid female parent) does not produce seeds. The hybrid is a triploid (3X=33), and is female and male sterile. Triploid plants have three sets of chromosomes, and three sets cannot be divided evenly when they go into two daughter cells during meiosis (the cell division process that produces the gametes). Since the triploid hybrid is female sterile, the fruit are seedless. Because the triploid is also male sterile, it is necessary to plant a diploid cultivar in the production field to provide the pollen that stimulates fruit to form. Usually, one third of the plants in the field are diploid and two thirds are triploid. Cultivars should be chosen that can be distinguished easily so the seeded diploid fruit can be separated from the seedless triploid fruit for marketing.

Breeders interested in the production of seedless triploid hybrids need to develop tetraploid inbred lines to be used as the female parent in a cross with a diploid male parent. One of the major limiting steps in breeding seedless watermelons is the small number of tetraploid inbreds available. Development of seedless hybrids will be discussed in the following stages: 1) choice of diploid lines, 2) production of tetraploid plants, 3) tetraploid line development, and 4) hybrid production and testing.

Stage 1 involves choice of diploid lines to use in tetraploid production. Most of the tetraploid lines being used by the seed industry have gray rind so that, when crossed with a diploid line with striped rind, it will be easy to separate self-pollinated progeny (which will be seeded fruit from the female parent line) from cross-pollinated progeny (which will be seedless fruit from the triploid hybrid). The grower will want to discard the gray fruit so they are not marketed as seedless watermelons by mistake.

Stage 2 is the production of tetraploid plants. Many methods have been used effectively in other crops to produce polyploids, including tissue culture regeneration, temperature shock, and X-rays. However, colchicine (C22H25O6N), a product from the seeds and bulbs of Colchicum autumnale L., is probably the most widely-used chemical for induction of watermelon tetraploids. Colchicine inhibits spindle formation, and prevents separation of chromosomes at anaphase. Of all the methods of colchicine application, shoot apex treatment at the seedling stage was found most effective.

For the seedling treatment method, the diploid line of interest is planted in the greenhouse in flats (8x16 cells is a popular size) on heating pads set to keep the soil medium at 85°F for rapid and uniform germination. When the cotyledons first emerge from the soil, the growing point is treated with colchicine to stop chromosome division and result in a shoot with four sets of chromosomes rather than two. The colchicine solution is used at a concentration of 1% for small-seeded cultivars (Minilee, Mickylee, Sweet Princess), 1.5% for medium-size-seeded cultivars (Allsweet, Crimson Sweet, Peacock Striped, Sugar Baby), and 2% for large-seeded cultivars (Black Diamond, Charleston Gray, Congo, Dixielee, Klondike Striped Blue Ribbon, Northern Sweet). Colchicine is applied in morning and evening for three consecutive days to each seedling, using 1 drop on small- or medium-size-seeded cultivars and 2 drops on large-seeded cultivars. The treatment produces plants that are diploid, tetraploid, or aneuploid, so it is necessary to identify and select the tetraploids in later stages. Treatment of the T0 diploids with colchicine results in about 1% of the seedlings (referred to as T1 generation tetraploids) being tetraploids. Some diploid cultivars and breeding lines produce a higher percentage of tetraploids than others.

Tetraploids can be detected by the direct method of counting chromosomes of cells under the microscope, or by comparing stem, leaf, flower, and pollen size with diploid controls. A popular method involves counting the number of chloroplasts in stomatal guard cells using a leaf peel under the microscope. Tetraploids have approximately 10-14 chloroplasts in each side of the guard cell (20-28 total), whereas diploids have only 5-6 in each side (10-12 total). The method is useful for screening many plants for ploidy level in the seedling stage before transplanting to the main part of the greenhouse or field nursery for self-pollination. Usually, multiple methods are used, identifying tetraploid seedlings using their phenotype in flats before transplanting, the chloroplast number in the stomatal guard cells of the true leaves in seedling flats and greenhouse pots, and by the appearance of the fruit and seeds at harvest after self-pollination in the greenhouse. Tetraploids usually have thicker leaves, slower growth, and shorter stems than diploids.

Stage 3 involves tetraploid line development. Tetraploid plants are selected (using methods such as leaf guard cell chloroplast number) in the T1 generation (plants from colchicine treated diploids) from the greenhouse flats where they were treated with colchicine. It is then necessary to plant the T2 generation in flats to verify that the plants are tetraploids in that next generation, and transplant the selections to greenhouse pots for self-pollination. Seeds from those selections can then be increased in larger plantings such as field isolation blocks to get sufficient numbers of seeds per tetraploid line to use in triploid hybrid production.

The fertility and seed yield of tetraploid lines will increase over generations of self- or sib-pollination, probably because plants with chromosome anomalies are eliminated, resulting in a tetraploid line with balanced chromosomes and regular formation of 11 quadrivalents. Seed yield of tetraploid lines in early generations is often only 50-100 seeds per fruit (vs. 200-800 for diploids). Another problem with raw tetraploids is poor seed germination, making it difficult to establish uniform field plantings. It may require as much as 10 years of self-pollination before sufficient seeds of tetraploid lines can be produced for commercial production of triploid hybrids. Advanced generations of tetraploid lines usually have improved fertility, seed yield, and germination rate compared to the original lines. Commercial seed production of a triploid hybrid cultivar requires 60 kg to meet market needs, with approximately 220 tetraploid plants per kg of triploid seeds produced.

Stage 4 is the evaluation of tetraploids (usually T4 generation or later) as parents of triploid hybrids. The tetraploids should be evaluated directly for gray rind pattern, high seed yield, and other traits such as male sterility for reduced hand labor in hybrid seed production. The major test for tetraploids however, is as female parents in triploid hybrid seed production after making controlled crosses using diploid male parents. The resulting hybrids are tested in yield trials with two rows of triploid plots alternating with one row of diploid plots to assure adequate pollen for fruit set in the triploid hybrids. Useful tetraploid inbreds should produce triploid hybrids with excellent yield and quality for the market type and production area of interest.

Triploid Evaluation

Evaluation of triploid hybrids is similar to evaluation of diploid cultivars already discussed previously. There are a few special considerations, however. Triploids are not inherently superior to diploids, so triploid hybrids can be better or worse than their diploid parental lines. Therefore, as in the case of diploid hybrids, many combinations of parental lines should be evaluated in triploid yield trials to identify the ones producing hybrids with the best performance. In general, diploid inbred parents that have poor horticultural performance will produce triploid hybrids having poor performance.

One problem affecting triploid hybrids is empty seed coats (colored or white) in the fruit. Under some environmental conditions, fruit are produced with large obvious seed coats that are objectionable to consumers. Triploids should be tested for seed coat problems in the fruit during trialing. Some selection should also be done on the parents before triploid production. Seed coats will be large in the hybrids if the parents have large seeds. Seed size is genetically controlled, with at least three genes involved: l, s, and tss. Besides genetic effects, certain environmental conditions seem to increase the number of hard seed coats in triploid hybrids.

Commercial production of elite triploid hybrids is done by hand in locations where labor is inexpensive, or by bee pollination in isolation blocks. The tetraploid and diploid inbreds are planted together in alternating rows, or in alternating hills within each row. Where labor is cheap, the staminate flowers can be collected from the male (diploid) parent and used to pollinate the pistillate flowers on the female (tetraploid) parent. Pollinated flowers should be capped the previous day to keep bees out, then covered after pollination to prevent self or sib-pollination after the cross has been made. The flowers should be tagged with the date so that the fruit can be harvested 28 days later.

A method that requires less hand labor is to plant the male and female parents in alternating rows, and to remove all staminate flowers from the female parent rows during peak flowering time, usually a period lasting several weeks. Pistillate flowers on the female parent are tagged on the day they open with the date to assure that the fruit are mature when harvested, and to harvest only fruit that were pollinated during the time staminate flowers were removed from the female parent. Seeds that are harvested can also be sorted mechanically for size, weight or density to separate triploid seeds (resulting from cross pollination) from tetraploid seeds (resulting from self- and sib-pollination).

When seed production is by bee pollination in isolation blocks, the triploid flowers are sib- or cross-pollinated 84% of the time, producing 3X and 4X seeds (progeny). If the 2X and 4X parents of the 3X hybrid have different rind patterns, each of the three ploidy levels can be distinguished at harvest. A useful combination is for the tetraploid parent to have fruit with gray rind, the diploid parent to have fruit with wide stripes, so the resulting triploid hybrid will striped fruit, easily distinguished from the gray-fruited tetraploids that result from self- or sib-pollination of the female parent.