The mitotic inhibitors, colchicine and oryzalin, were evaluated for their effects on callus, adventitious shoot formation, and tetraploid induction of Euphorbia pulchurrima `Winter Rose'. In vitro grown leaf sections were placed on various media supplemented with either colchicine or oryzalin at various concentrations for 1 to 4 days. Colchicine was less damaging to leaf tissues than oryzalin. On various colchicine-containing media, prolific calluses were produced and adventitious shoot formation was observed. Regenerated shoots were found to be diploid as determined by flow cytometry. On media supplemented with oryzalin (28.9 μm to 144 μm), leaf tissues produced callus but failed to form adventitious shoots. Samples of calluses produced on oryzalin-containing media were subject to analysis using flow cytometry and were found to be diploid. These results suggest that the colchicine is less toxic on poinsettia tissues and shoot induction than oryzalin. Additional experiments are needed to establish a protocol for in vitro induction of poinsettia tetraploid with colchicine and oryzalin.
Kimberly A. Pickens, (Max) Z.-M. Cheng and Stephen A. Kania
Jose Lopez-Medina, James N. Moore and Ronald W. McNew
Inheritance of the primocane-fruiting (PF) characteristic was studied in seedling populations of tetraploid (4x) blackberries (Rubus subgenus Rubus). Four selections (A-1836, A-593, A-830, and A-1680) and two cultivars (`Arapaho' and `Shawnee') were used as parents in a full diallel crossing scheme. Selection A-593 was used as the main source for PF due to its origin (`Brazos' × `Hillquist,' the latter an old PF cultivar). All parents except `Shawnee' have A-593 in their parentage; among the parents, only A-1836 fully expresses PF. Selfing of A-1836 resulted in 100% PF offspring, indicating that A-1836 is homozygous for this trait. Selfing of A-593, A-830, and `Arapaho' produced either a 35:1 or a 20.8:1 FF (floricane or summer-fruiting):PF segregation ratio, fitting a tetrasomic inheritance model under either random chromosome assortment (RCSA) or random chromatid assortment (RCTA), respectively, also suggesting that PF is controlled by a single recessive gene and that the parents are duplex (AAaa) for this trait. Selection A-1680 and `Shawnee' selfed did not produce PF progeny, but when crossed with the nulliplex A-1836, gave a 27:1 FF:PF ratio, indicating RCTA and that they are triplex (AAAa) for PF. According to these research, both gametic outputs (RCSA and RCTA) seem to operate in 4x blackberry. The intensity in expression of PF had a negative relationship with time to harvest, with those seedlings showing the highest PF scores producing a crop in early to mid-August. This knowledge will be helpful in implementing breeding strategies to produce PF blackberry cultivars.
Michael E. Compton and D.J. Gray
Adventitious shoots were obtained from watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai] cotyledons incubated on a modified Murashige and Skoog medium containing BA. Initial experiments comparing the effects of BA (0, 5, 10, or 20 μm) and IA4 (0, 0.5, or 5 μm) demonstrated that BA was required for adventitious shoot formation but its concentration in the medium was not critical. The addition of IAA to medium with BA increased callus production and inhibited shoot formation. However, the percentage of responding explants in the best treatment was <30%. Therefore, the manner in which cotyledon explants were prepared and seedling age at the time of explantation was examined to improve the organogenic response. The percentage of explants with shoots was improved by using explants that consisted of cotyledon bases (43%) or cotyledons cut in half longitudinally (39%). A lower percentage (16%) of cotyledons cut longitudinally into four pieces produced shoots. Explants taken from the apical half of cotyledons failed to regenerate shoots. Shoot formation was improved further by using explants from young seedlings. The percentage of explants with shoots was >90% for `Minilee', 64% for S86NE, and 50% for `Jubilee II' when explants were prepared from 5-day-old seedlings. Explants from nongerminated embryos or seedlings germinated for 10, 15, or 20 days produced fewer shoots. The effect of several cytokinins on shoot organogenesis was then examined using the optimized protocol. The percentage of explants with shoots and the number of shoots per explant were about two to four times higher when 5 to 10 μm BA was used compared to the most effective kinetin (20 μm) or thidiazuron (0.1 μm) concentration. The percentage of explants with shoots and the number of shoots per explant were greater for diploid (57% and 2.2, respectively) than for triploid (22% and 0.6, respectively) or tetraploid (20% and 0.8, respectively) lines. Chemical names used: N -(phenylmethyl)-1 H -purin-6-amine (BA); 6-furfurylaminopurine (kinetin); N -phenyl-N' -1,2,3-thiadiazol-5-ylurea (thidiazuron); 1 H -indole3-acetic acid (IAA).
Ninety-eight percent of cotyledons of Cucumis metuliferus (PI 482439) regenerated shoot buds in 5 weeks on MS medium with 10 μm BA. Regeneration rates on agar gelled medium and liquid/membrane system were compared after weekly transfers of tissue from liquid/membrane to agar during the 5 weeks of regeneration culture. Number of shoots and buds increased from six (on agargelled medium) to nine per cotyledon when explant on liquid/membrane system for 3 or 4 weeks was transferred to agar gelled medium. Shoot development, rooting, and survival in greenhouse was adequate regardless of whether regeneration was initiated on agar or liquid/membrane system. Tetraploid regenerants were found to be about 9% of the almost 400 regenerants screened. Pollen morphology was a quick and definitive screening technique for tetraploid plants. Frequency of tetraploid plants was similar after 5 weeks of induction on either agar or liquid/membrane system. This frequency decreased by a factor of 10 following transfer of explants from membrane to agar after the first week of induction. Timing of this transfer plays a critical role in eliminating tetraploid variants.
Eliezer S. Louzada, Jude W. Grosseti, Frederick G. Gmitter Jr., Beatriz Nielsen, J.L. Chandler, Xiu Xin Deng and Nicasio Tusa
Protoplast culture following polyethylene glycol-induced fusion resulted in the regeneration of vigorous tetraploid somatic hybrid plants from eight complementary parental rootstock combinations: Citrus reticulata Blanco (Cleopatra mandarin) + C. aurantium L. (sour orange), C. reticulata (Cleopatra mandarin) + C. jambhiri Lush (rough lemon), C. reticulata (Cleopatra mandarin) + C. volkameriana Ten. & Pasq. (Volkamer lemon), C. reticulata (Cleopatra mandarin) + C. limonia Osb. (Rang-pur), C. sinensis (L.) Osb. (Hamlin sweet orange) + C. limonia (Rangpur), C. aurantium (sour orange) + C. volkameriana (Volkamer lemon) zygotic seedling, C. auruntium hybrid (Smooth Flat Seville) + C. jambhiri (rough lemon), and C. sinensis (Valencia sweet orange) + Carrizo citrange [C. paradisi Macf. × Poncirus trifoliata (L.) Raf.]. Diploid plants were regenerated from nonfused callus-derived protoplasts of Valencia sweet orange and Smooth Flat Seville and from nonfused leaf protoplasts of sour orange, Rangpur, rough lemon, and Volkamer lemon. Regenerated plants were classified according to leaf morphology, chromosome number, and leaf isozyme profiles. All somatic hybrid plants were tetraploid (2n = 4× = 36). One autotetraploid plant of the Volkamer lemon zygotic was recovered, apparently resulting from a homokaryotic fusion. These eight new citrus somatic hybrids have been propagated and entered into field trials.
J.P. Syvertsen, J.W Grosser and L.S. Lee
We grew three diploid (2X) Citrus rootstock seedlings and their autotetraploids (4X) at elevated CO2 to obtain insights into limitations on growth and net gas exchange that have been associated with tetraploidy. Well-nourished Volkamer lemon (Volk), Troyer citrange (Troy), and Cleopatra mandarin (Cleo) were grown in greenhouses at ambient or twice ambient CO2 for 3 months. We measured plant growth, water relations, mineral nutrition, and net gas exchange characteristics of leaves. Overall, tetraploid roots were thicker as 4X had lower root length: dry weight ratio or specific root length (SRL) than 2X roots. Tetraploid plants were smaller and had higher root/shoot ratios, shorter fibrous roots, and lower whole plant transpiration than 2X. Tetraploids also had lower leaf N and P concentrations on a dry weight basis. Since 4X leaves had thicker leaves (more dry weight per area) than 2X leaves, these nutrient differences disappeared when expressed on an leaf area basis. Elevated CO2 increased plant growth but decreased leaf N, P, and K apparently by a growth dilution effect. Elevated CO2 also increased fibrous root thickness, leaf thickness, and net assimilation of CO2 (ACO2) but decreased stomatal conductance and transpiration such that leaf water use efficiency increased. There was no effect of ploidy level on ACO2 but 4X Volk and Troy had lower rates of ACO2 than their diploids at elevated CO2. Hydraulic conductivity of intact root systems (measured in a pressure pot) was correlated to total plant growth but variability obscured effects of CO2 or ploidy on root conductivity. The low SRL of tetraploids were correlated with lower rates of water use and lower leaf nutrient concentrations, which may be operative in determining the growth characteristics associated with tetraploidy.
Perry E. Nugent and Dennis T. Ray
Since 1968, three spontaneous 4× melons (Cucumis melo L.) plants were discovered in our field or greenhouse plantings. Two were found in the cultivar Planters Jumbo and one in the virescent marker C879-52. Each of these 4× plants had rounded cotyledons, shorter internodes, thicker stems and leaves, more hairs, and smaller fruits, with larger stem and blossom scars, than their 2× counterparts. Also, their flowers, pollen grains, stomates, and seeds were larger. The discovery of a 4× virescent plant in 1987 allows easier germplasm transfer between ploidy levels. Morphological characteristics of 2× and 4× melons will allow identification without need for chromosome counts.
J.P. Syvertsen, L.S. Lee and J.W Grosser
Diploid (2x) and autotetraploid (4x) Citrus L. rootstock cultivars were grown at elevated CO2 to obtain insights into limitations on growth and net gas exchange that have been associated with tetraploidy. Well-nourished 2x and 4x seedlings of `Volkamer' lemon (Volk, C. volkameriana Ten & Pasq.), `Troyer' citrange [Troy, C. sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] and `Cleopatra' mandarin (Cleo, C. reticulata Blanco.), were grown in greenhouses at either ambient or twice ambient CO2 for 4 months. Plant growth, water relations, mineral nutrition, and net gas exchange characteristics of leaves were measured. Most 4x plants were smaller and had lower rates of whole plant transpiration but shorter fibrous roots than 2x plants. Fibrous roots of 4x were thicker than 2x roots as indicated by a lower specific root length (SRL) in 4x than in 2x roots. Root hydraulic conductivity was correlated to total plant growth but there were no effects of CO2 or ploidy on root conductivity. Tetraploid leaves had lower N concentrations than 2x leaves when expressed on a dry weight basis but these differences disappeared when N concentration was expressed on an leaf area basis because 4x leaves had more leaf dry weight per area (LDW/a) than 2x leaves. Plant growth was greater and SRL was lower at elevated CO2 than at ambient CO2. LDW concentrations of N, P, and K were lower at elevated CO2 than at ambient apparently due to a growth dilution effect. LDW/a, net CO2 assimilation (ACO2), and leaf water use efficiency were greater at elevated CO2 than at ambient. Overall, there was no effect of ploidy on ACO2 but 4x Volk and Troy had lower rates of ACO2 than their 2x at elevated CO2. Net gas exchange of tetraploid leaves was less responsive to elevated CO2 than 2x leaves. The low SRL of tetraploids was correlated with low whole plant transpiration rates and low leaf area-based N concentrations, which may be operative in determining the growth characteristics associated with tetraploidy.
Nicholi Vorsa and Richard Novy
Vaccinium darrowi (D) is a wild blueberry species with low chilling requirements for budbreak, and heat and drought tolerance. Breeding efforts to incorporate these desirable traits into cultivated blueberry (V. corymbosum) (C) would be facilitated with a better understanding of the genomic homology between the two species. An interspecific tetraploid hybrid (CCDD, 2n=4x=48) was used to evaluate genome homology and interspecific recombination. Pollen mother cells examined at diakinesis and early metaphase I exhibited an average of 4.6 chain bivalents, 11.4 ring bivalents, 1.0 chain quadrivalent, and 3.0 ring quadrivalents. This data most closely fits a chromosome pairing model in which there is a greater pairing affinity between homologues than homoeologues. An analysis of the inheritance of 14 RAPD markers unique to V. darrowi in 72 backcross progeny of the V. darrowi–corymbosum hybrid also supported the pairing model: Seven of the 14 markers deviated significantly from tetrasomic inheritance ratios, expected if chromosome pairing was totally random. On the basis of the cytogenetic and RAPD analyses, the genomes of V. darrowi and V. corymbosum are divergent from one another, with preferential pairing within genomes. This outcome suggests there may be difficulty in breaking undesirable linkages when introgressing desirable traits from V. darrowi to V. corymbosum.
Jeffrey Adelberg, Maria Delgado and Jeffery Tomkins
Two tetraploid and two diploid varieties of daylily were micropropagated on a shaker in MS liquid medium containing high and low sugar levels (3% and 6% sucrose), 2 BA levels (0.32 and 3.2 μm), at two densities (57 and 171 explants/L), in the presence (0.32 μm) and absence of ancymidol. Biomass and media use were partitioned for the four genotypes and 32 cultural conditions with three replications (4 × 2 × 2 × 2 × 2 × 3). Genotype greatly effected f resh weight, dry weight, media, sugar and water use, but ploidy had little effect. Vessels at high density (171 explants/L) produced 1.8× more fresh weight, 1.4× more dry weight, used 1.6× more media and sugar than low density (57 explants/L). Plants from low density were 1.7× larger, 2× greater dry weight, and used 2× more sugar and media, than from high-density culture (per explant). Doubling the initial sugar level increased dry weight and sugar use 1.3×. There was a linear relation between sugar residual and percentage of dry weight (R 2 = 0.55, P < 0.0001), where a 1% increase in °Brix raised percentage of dry weight 1.8 units over the range of 9% to 22%. Ancymidol and BA had less effect on plant size, sugar and media use than genotype or plant density. Greenhouse survival was reduced by including ancymidol (90% to 30%) and increased BA concentration (85% to 35%). Lab plant density and initial sugar concentration had no apparent effect on greenhouse growth. `Barbara Mitchell' had greatest mass, used more sugar and media than the other varieties, yet had least greenhouse growth. Nutrient use with `Barbara Mitchell' was linearly correlated (R 2>80%) to lab growth for seven of 12 ions. P and Fe supply was inadequate to support optimal growth, as indicated by low residual in media (>1% of MS formulation).