Previous research has optimized the colchicine dropper technique for chromosome doubling under greenhouse conditions. In recent years, in vitro germination of cut strawberry achenes has greatly increased germination rates. Combining the two techniques would be especially useful when chromosome doubling is desired for interspecific hybridization. Fragaria vesca was chosen for initial study. Treatments included colchicine levels of 0%, 1%, 2%, 3%, 4%, or 5% (w/v); exposure time to colchicine was from 6 to 16 to 26 hours; application was at the cotyledon stage or after the first true leaf formed; presence or absence of 3 g activated charcoal/liter; and presence or absence of DMSO. Media consisted of MS salts and vitamins, 30 g sucrose/liter, and 2.5 g phytogel/liter. Charcoal enhanced upward growth of seedlings, thus allowing better placement of colchicine droplets. Reduced exposure time and application at the first true-leaf stage allowed higher levels of colchicine to be used without greatly reducing the vigor of treated seedlings.
Parthenogenetic haploid embryos of `Crimson Sweet', `Halep Karasi', `Sugar Baby' and `Panonia F1' watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] were obtained after pollination with γ-irradiated (200 or 300 Gy) pollen. Some globular and heart-shaped embryos were observed in fruit harvested 2 to 5 weeks after pollination. The number of embryos per 100 seeds was highest for `Halep Karasi'. After in vitro culture, 17 haploid plants were obtained and doubled haploid lines were generated after chromosome doubling using colchicine.
Ploidy level and fertility status are often the two biggest barriers a breeder must overcome when trying to incorporate novel characteristics among related taxa. This study was aimed at developing an efficient chromosome doubling method for Buddleja L., commonly known as butterfly bush, with the goal of equalizing the ploidy level and restoring the fertility of a diploid (2n=38) F1 interspecific hybrid that has a unique orange color but happens to be sterile. This method would ease the crossing of the hybrid to the tetraploid (2n=76) B. davidii Franch. cultivars commonly found in the industry. An antimitotic treatment of oryzalin was tested on 02-25-142 (B. madagascarensis Lam. × B. crispa Benth.) in vitro using nodal sections. A factorial of varying concentrations [3, 5, and 7 μM (micromolar)] by different exposure times (1, 2, and 3 day) plus controls was set up. Oryzalin appeared to be an efficient agent for chromosome doubling in Buddleja. Significant differences in the number of polyploids were not seen between chemical concentrations and exposure times. However, higher chemical concentrations and exposure times did have a significant effect on the number of nodes that survived tissue culture. Increased leaf size and color, stem thickness, shortened internode length, and upright growth habit were all good early phenotypic indicators of polyploidy induction as later confirmed by flow cytometry. Significant increases in pollen viability accompanied chromosome doubling as crosses between 02-25-142 × B. davidii cultivars produced viable seedlings.
Spontaneous chromosome doubling occurred in shoot apices of two diploid (2n = 22) Hatiora ×graeseri Barthlott ex D. Hunt (Easter cactus) clones and yielded stable periclinal cytochimeras with a diploid epidermis and tetraploid subepidermis. The cytochimeras produced disomic gametes (n = 22) and displayed tetrasomic inheritance at polymorphic isozyme loci. Diploid clones were highly self-incompatible (SI) but both cytochimeras were self-compatible (SC). Analysis of pollen tube growth in selfed or outcrossed styles revealed that polyploidy altered the incompatibility phenotype of pollen without affecting the incompatibility phenotype of the pistil. Morphological data (guard cell length, stomatal density, and pollen diameter), segregation ratios at isozyme loci, and fruit/seed yields indicate that S1 progeny are SC, nonchimeral, and tetraploid. Breakdown of the SI system in the cytochimeras was attributed to formation of compatible heteroallelic pollen. These results provide a rational explanation for the correlation between ploidy level and breeding behavior in cacti. Production of SC autotetraploid clones from SI diploids by chromosome doubling may be useful in development of cacti as fruit crops.
A synthetic autotetraploid derived by colchicine treatment of a Vaccinium elliottii Chapm. plant (2n = 2x = 24) was used to study the effect of chromosome doubling on the ability of this noncultivated species to cross with the cultivated tetraploid highbush blueberry (V. corymbosum L.). Mean pollen germination was 28%1 for the autotetraploid plant, compared to 53% for the diploid V. elliottii plant. However, the number of seedlings obtained per flower pollinated on the tetraploid highbush cultivar O'Neal rose from 0.01 when diploid V. elliottii was the pollen source to 3.86 when pollen from the autotetraploid V. elliottii plant was used. Reciprocal crosses between diploid V. elliottii and its autotetraploid and selfs of the autotetraploid produced no seedlings. Meiotic irregularities, such as multivalent during metaphase, laggards, and unequal chromosome disjunction, were observed in the autotetraploid, but most chromosomes were associated as bivalents.
Homozygous doubled haploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions could be useful to accelerate breeding for resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollinating with pollen irradiated with a Co60 γ-ray source at 500 Gy; 2) in vitro rescue of putative parthenogenic embryos identified by their morphology and localized using a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by flow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfing of selected lines. Codominant markers and flow cytometry confirmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μm colchicine. Rescue of diploid or chimeric plants was shown by flow cytometry, prior to their acclimation and planting in the greenhouse. Selfing of colchicine-treated haploid plants allowed for the perpetuation by seed of homozygous lines. The high rate of seed set, 90% of the lines produced seed, facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation. Breeding for new commercial hybrid cultivars will be accelerated. DHLs are ideal resources for genomic analyses.
Alstroemeria, also known as Lily-of-the-Incas, Inca Lily, or Peruvian Lily, has been bred at the Univ. of Connecticut since 1985. In vitro procedures have been integrated with traditional breeding techniques to create new and exciting cultivars. Embryo culture has been used to generate interspecific, intraspecific, and intergeneric hybrids that would not have been possible with traditional breeding. Somaclonal variation has been used to create new plants from spontaneous and induced mutations, but, in most cases, the plants have not been acceptable commercially. Chromosome doubling with colchicine has been used for fertility restoration of sterile diploids. Somatic embryogenesis has also been studied quite extensively; somatic embryos are easily obtained from zygotic embryos of Alstroemeria. In vitro fertilization procedures are currently being studied in order to hasten embryo development after hybridization has occurred. Because Alstroemeria plants are slow to propagate by traditional rhizome division, micropropagation is used to multiply new cultivars rapidly. Because the production of pathogen-free plants is one of the goals of our breeding and new plant introduction programs, meristem culture and thermotherapy are also being studied. All of these techniques will be described during the workshop.
An objective of our rose breeding research is to transfer resistance to blackspot and other diseases from wild diploid species to modern rose cultivars. Interspecific hybrids among blackspot-resistant diploid species were chosen for chromosome doubling to produce fertile amphidiploids that could be hybridized to the tetraploid commercial germplasm. Five such F1 interspecific hybrids were treated with colchicine. The study included two different application procedures (shake in colchicine solution or colchicine in media), four colchicine concentrations (0.05%, 0.1%, 0.15%, and 0.20%), and five treatment periods (1, 3, 5, 8, and 10 days). After colchicine treatment, all the materials were cultured in vitro. One thousand-thirty-seven surviving explants were selected for typical “gigas” characteristics of doubled diploids. Chromosome counts on shoot tips of these selected genotypes confirmed 15 amphidiploids. The best colchicine treatment varied among the interspecific hybrids. Higher colchicine concentrations or duration reduced growth rating, rooting, and percent survival. The recognition of amphiploids and ploidy chimeras from young seedlings will also be discussed.
The use of wild species as breeding materials was tried for expanding hereditary variation in strawberry. Some interspecific hybrids setting large fruits with peculiar aroma have been bred by pollination of F. vesca to F. xananassa. Although Asian wild diploid strawberries such as F. nilgerrensis or F. iinumae have not been exploited as a breeding material until the present, the crossing test between cultivated strawberries (8x) and the Asian wild strawberries (2x) were attempted. The interspecific hybrids originated from pollination of F. nilgerrensis or F. iinumae to F. xananassa cv. `Toyonoka' were all sterile pentaploids. By in vitro colchicine treatment of these sterile hybrids for chromosome doubling, many fruiting interspecific hybrids were produced. In particular, some superior hybrids were obtained from `Toyonoka' × F. nilgerrensis. From the results of RAPD analysis, the interspecific hybrids had the fragments specific for both parents. While their morphological characters were close to `Toyonoka', they had some characters from F. nilgerrensis, such as numerous hair on their petioles and peduncles. Their fruits have good characters that are same level of cultivated strawberry about size, Brix, acidity, and vitamin C content. The flesh is soft and skin color is pale pink. The aroma components are resemble F. nilgerrensis, and enrich ethyl acetate. The fragrance of interspecific hybrid like peach is characteristic.
Poinsettia, Euphorbia pulcherrima, is an important holiday symbol and is the number one flowering potted plant in the United States. The technique of chromosome doubling has been utilized to increase size of flowers, stems, and leaves of many species, and has been used in poinsettia breeding to obtain new cultivars. Application of colchicine or oryzalin to in vitro tissues may be used to enlarge the inflorescences and brackets and reduce the height of `Winter Rose'™ poinsettias, reduce the likelihood of chimeric tetraploids, and provide a rapid means for producing many tetraploid plants. The purpose of this research was to evaluate the effect of colchicine and oryzalin on callus and adventitious shoot formation of `Winter Rose'™ poinsettia with in vitro grown leaf tissues and its potential for tetraploid induction. In vitro grown leaf midvein sections were placed on various media supplemented with either colchicine or oryzalin at various concentrations for 1–4 days. Colchicine was least damaging to leaf tissues at concentrations of 0.25 or 250.4 μm. A large amount of callus, as well as adventitious shoots, were produced. Regenerated shoots were found to be diploid, determined by flow cytometry. On media with oryzalin (28.9–144 μm), leaf tissues produced callus, but not adventitious shoots. Calluses produced on oryzalin-containing media were tested using the flow cytometer and were found to be diploid.