Tetraploid Production of Seedless Watermelon | India

After reading this article you will learn about the tetraploid production of seedless watermelon.

Triploid hybrids produce seedless fruit. The tetraploid method for seedless watermelon production was invented by H. Kihara in Japan and these were available in 1951. He began development of tetraploids in 1939.

Development of triploid watermelons faces problems of 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 vigour for the grower; and the necessity for the diploid pollinizer to use up to one-third of the grower’s production field.

Seedless cultivars 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 resulting 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 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, although successful production has been observed with as little as 20% diploids. Cultivars 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.

Development of seedless triploid hybrids needs tetraploid inbred lines to be used as the female parent in a cross with a diploid male parent. Availability of small number of tetraploid inbreds is the major difficulty.

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 have seeded fruit from the female parent line) from cross-pollinated progeny (which will have 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. In watermelon, tetraploids can be produced routinely using plants regenerated from tissue culture or using the herbicide oryzalin.

Colchicine (C₂₂H₂₅O₆N), a poisonous alkaloid used in the treatment of gout, from the seeds and bulbs of Colchicum autumnale is a widely used chemical in watermelon for tetraploid production. 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 is 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 0.1% for small- seed size cultivars (‘Minilee’, ‘Mickylee’, ‘Sweet Princess’), 0.15-0.2% for medium-seed size cultivars (‘Allsweet’, ‘Crimson Sweet’, ‘Peacock Striped’, ‘Sugar Baby’), and 0.2-0.5% for large-seed size cultivars (‘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 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. 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 stomata), 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 (1%) 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.

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 labour 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.

Evaluation of triploid hybrids is similar to evaluation of diploid cultivars. 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 (coloured 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: 1, s, and ts. 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 Seed Production of Triploid Watermelon:

1. Through Manual Pollination:

In this method tetraploid and diploid lines are planted in alternate rows or in alternating hills within each row. Female buds are capped in evening. Next morning, freshly opened staminate flowers are collected from diploid male parent and are used to pollinate the pistillate flowers bagged/covered previous evening.

Again the pollinated flowers are covered to prevent self or sib-pollination. The flowers should be tagged with the date so that the fruit can be harvested after 40-50 days.

2. Pollination through Bees in Isolation Block:

In this method, tetraploid (seed parent) and diploid (pollen parent) are planted in alternate rows in isolation block. During flowering, all staminate flowers from seed parent are removed for a period lasting several weeks. Pistillate flowers on female (seed parent/tetraploid line) are tagged with date to ensure their harvesting after 40-50 days after anthesis.