Seedless Variety Development
Tetraploid Production. Use of triploid hybrids has provided a method for production of seedless fruit. The tetraploid method for seedless watermelon production was invented by H. Kihara. He began development of tetraploids in 1939, and had commercial triploid hybrids available 12 years later. The development of triploid varieties adds several problems to the process of watermelon breeding: extra time for the development of tetraploids; additional selection against sterility and fruit abnormalities in tetraploid lines; choice of parents for low incidence of hard seed coats in the hybrids; the reduction in seed yield per acre; reduced seed vigor for the grower; and the necessity for the diploid pollenizer to use up to one-third of the grower’s production field.
Seedless varieties are produced by crossing a tetraploid (2n=4x=44) inbred line as the female parent with a diploid (2n=2x=22) inbred line as the male parent of the hybrid. The reciprocal cross (diploid female parent) does not produce seeds. The resulting hybrid is a triploid (2n=3x=33). Triploid plants have three sets of chromosomes, and three sets cannot be divided evenly during meiosis (the cell division process that produces the gametes). This results in non-functional female and male gametes although the flowers appear normal. Since the triploid hybrid is female sterile, the fruit induced by pollination tend to be seedless. Unfortunately, the triploid has no viable pollen, so it is necessary to plant a diploid variety 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, although successful production has been observed with as little as 20% diploids. Varieties should be chosen that can be distinguished easily so the seeded diploid fruit can be separated from the seedless triploid fruit for harvesting and 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 should 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. In watermelon, tetraploids can be produced routinely using plants regenerated from tissue culture or using the herbicide oryzalin. Colchicine (C22H25O6N) is an alkaloid used in the treatment of gout. It is taken from the seeds and bulbs of Colchicum autumnale and is a widely-used method for tetraploid production in watermelon. 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 (8×16 cells is a popular size) on heating pads that 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 produce a tetraploid shoot with four sets of chromosomes rather than two. The colchicine solution is used at a concentration of 1% for small-seed size varieties (‘Minilee’, ‘Mickylee’, ‘Sweet Princess’), 1.5-2.0% for medium-seed size varieties (‘Allsweet’, ‘Crimson Sweet’, ‘Peacock Striped’, ‘Sugar Baby’), and 2-5% for large-seed size varieties (‘Black Diamond’, ‘Charleston Gray’, ‘Congo’, ‘Dixielee’, ‘Klondike Striped Blue Ribbon’, ‘Northern Sweet’). Colchicine is applied to the seedling growing point in the morning and evening for 3 consecutive days, using 1 drop on small- or medium-seed size plants and 2 drops on large-seed size varieties. Protect workers who are handling colchicine with safety equipment such as gloves. 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 varieties and breeding lines produce a higher percentage of tetraploids than others. For example, ‘Early Canada’ produces many tetraploids and ‘Sweet Princess’ does not.
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 guard cell (20-28 total on both sides of the stomate), whereas diploids have only 5-6 in each guard cell (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 T0 generation (plants from colchicine treated diploids) from the greenhouse flats where they were treated with colchicine. It is then necessary to plant the T1 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 (T2) 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 chromosome number and regular formation of 11 quadrivalents. Seed yield of tetraploid lines in early generations is often only 50-100 seeds per fruit and sometimes as low as 0-5 seeds compared to 200-800 seeds for diploids. Another problem with early generation 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. Some companies require more than 100 lbs. of seed of a tetraploid inbred to be available before beginning commercial production of the triploid hybrid variety. Approximately 110 tetraploid plants are required for production of each pound of triploid seeds.
Stage 4 is the evaluation of tetraploids (usually T3 generation or later) as parents of triploid hybrids. The tetraploids should be evaluated directly for 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.
Note that colchicine is used to convert diploids (T0) into tetraploids (T1). Thus, the chemical only contacts the growing point of the converted (T1) plants. Several generations later, the tetraploids with improved seed yield (T3) are used to produce seeds of triploids after pollination of tetraploid (female parent) with diploid (male parent) inbreds. No colchicine comes into contact with the seedless watermelon cultivar (triploid), or its female (tetraploid) or male (diploid) parents.
Triploid Evaluation. Evaluation of triploid hybrids is similar to evaluation of diploid varieties already discussed. 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. Triploid fruit should be evaluated for seed coat problems 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. Use of tetraploid lines with small or tomato-size seeds may help solve the problem. Besides genetic effects, certain unknown environmental conditions seem to increase the number of hard seed coats in poor performing triploid hybrids.
Commercial production of elite triploid hybrid seed 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 abundant, 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 35-50 days later.
A method that requires less hand labor is to plant the pollen and seed parents in alternating rows, and to remove all staminate flowers from the seed parent rows during 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 tetraploid 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. For safety, the pollen parent plants should be destroyed after fruit are set on the seed parent plants. A useful combination is for the tetraploid parent to have fruit with a gray rind pattern, and the diploid parent to have fruit with wide stripes, so the resulting triploid hybrid will have striped fruit, easily distinguished from the gray fruited tetraploids that result from self- or sib-pollination of the female parent.