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Acid Citrus Fruit Cultivar Improvement Via Interploid Hybridization

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Acid Citrus Fruit Cultivar Improvement Via Interploid Hybridization Reprinted from Proc. Fla. State Hort. Soc. 113:124-126. 2000. ACID CITRUS FRUIT CULTIVAR IMPROVEMENT VIA INTERPLOID HYBRIDIZATION J. L. CHANDLER, Z. VILORIA AND J. W. GROSSER crosses. The lemon cultivars, somatic hybrids, and cross combina- University of Florida, IFAS tions are specified in Tables 1 and 2. Citrus Research and Education Center Flowers from seed parents were emasculated before anthesis 700 Experiment Station Road and immediately pollinated with stored pollen collected in the same Lake Alfred, FL 33850 season. Approximately three months after pollination, the fruits were harvested and stored temporarily at 4°C. Fruits were surface Additional index words. Lemon, lime, seedlessness, somatic sterilized for at least 30 min. with a solution of 20% bleach (5.25% hybrid, triploid. NaOCl) with three drops of liquid detergent added as a surfactant. Thereafter, the fruits were cut at the equatorial region without dam- Abstract. We have conducted several interploid crosses be- aging the core where the seeds are embedded. After twisting and tween fertile somatic hybrids such as ‘Hamlin’ + ‘Femminello’, separating the two halves, seeds were extracted and the embryos ‘Key’ lime + ‘Valencia’, ‘Valencia’ + ‘Femminello’, ‘Milam’ + carefully excised under a stereo microscope. Embryos in globular, ‘Femminello’ and 4X ‘Femminello’ and various diploid acid- heart and cotyledon stages were cultured on EME medium (Grosser fruit cultivars to generate seedless triploids with horticultural- and Gmitter, 1990) under continuous light at 25 to 27°C. ly acceptable fruit and improved cold-hardiness (inherited One month later, the germinated embryos were transferred onto from sweet orange), suitable for production in sub-tropical re- RMAN (rooting medium) (Grosser and Gmitter, 1990) for about gions. Embryo rescue was utilized to recover triploid progeny one month. Embryo germination was induced in Petri dishes (100 ´ about three months after pollination. Embryos in different de- 15 mm) and resulting plantlets were grown in Magenta vessels. velopmental stages were grown on Murashige and Tucker me- dium supplemented with 500 mg/L malt extract. A total of fifty Seedlings were transplanted into 38-well trays contained a commer- three triploids and twenty two tetraploids were recovered from cial soil mixture (Metro Mix-500®). After acclimatization at 80% different cross combinations. RH at 28°C, the plants were moved into a greenhouse under normal conditions. Lemons and limes are not commercially grown in central Flor- ida due to their lack of cold hardiness and their adverse horticultur- Ploidy Analysis al responses to the semi-tropical environment. Typically, lemon fruits grow too large; and storage quality is negatively affected by Ploidy level of new seedlings was determined by cytology or peel condition and fungal diseases. ‘Key’ limes are better suited to flow cytometry analysis. The first technique implies direct micro- the warm, humid climate but have no tolerance to frost; also, they scopic counting of chromosomes from actively growing root tip are susceptible to lime anthracnose and citrus tristeza virus (CTV). cells. A modified hematoxylin staining protocol was applied as de- Lemons are not susceptible to CTV, and can tolerate slightly scribed by Grosser and Gmitter (1990). For the latter technique, the colder winter conditions; however, melanose and scab can compli- relative amount of nuclear DNA was determined by flow cytome- cate cultivation. Citrus canker poses a hazard to both cultivars. try. Ploidy determinations by flow cytometry were performed by Previously produced somatic hybrids that combine ‘Key’ lime the ICBR, Flow Cytometry Core, University of Florida, according and ‘Femminello’ lemon with ‘Valencia’ or ‘Hamlin’ sweet orang- to the procedure previously described by Arumuganathan and Ear- es are flowering and producing pleasant acid-type fruit (Grosser et le (1991) and using a FacStar PLUS flow cytometer (Becton-Dick- al., 1989; Tusa et al., 1990 and 1992). Although these somatic hy- inson, Franklin Lakes, NJ). brid trees are more cold-hardy than cultivated limes and lemons, their fruit is horticulturally inferior to the standard cultivated types. Results and Discussion Obtaining triploid hybrids will result in seedless fruit, reduced fruit size, and thinner rinds. These improvements should facilitate mar- Fruit set and the number of seed per fruit were affected by the keting. Improved cold-hardiness is expected from the use of sweet pollen parent (Table 1), with the highest number of seeds observed orange somatic hybrids in the breeding scheme. Using limequat in ‘Lisbon’ and ‘Todo del Año’. The percentage of empty seeds hybrids (which contain Fortunella genes) in the crosses could in- was higher than 25% in most of the diploid-by-tetraploid crosses, troduce canker resistance to the new hybrids. Resistance to anthra- except for ‘Todo del año’ by (‘Key’ lime + ‘Valencia’), which cnose could also be inherited by these new hybrids. showed 6.95% embryoless seeds. Fully developed seeds were col- lected from the ‘Lisbon’ by (‘Hamlin’+‘Femminello’) crosses; Materials and Methods nevertheless, there were no hybrid embryos recovered. This sug- gests a rapid growth of nucellar embryos and the subsequent death ‘Key’ lime (Citrus aurantifolia Swing.), five lemon [C. limon of the zygotic one. Histological studies showed that most of the cit- (L.) Burm. F.] cultivars, four allotetraploid somatic hybrids rus embryos from diploid (monoembryonic) by tetraploid crosses (Grosser et al., 1989; Tusa et al., 1990 and 1992) and one autotet- abort due to the degeneration or abnormal development of the 4X raploid grown at The Florida Citrus Arboretum-Division of Plant endosperm (Esen and Soost, 1973). Industry (Winter Haven, FL) or CREC (University of Florida, Regardless of the cross combination, the number of recovered Lake Alfred, FL) were selected as breeding parents for interploid plants was low considering the number of extracted seeds and the polyembryonic characteristic of the maternal parents. The number of triploid hybrids was variable. The frequency was determined by the cross combination to a certain extent, and the in vitro germina- Florida Agricultural Experiment Station Journal Series No. N-01989. tion capacity of the embryos. The number of zygotic embryos was 124 Proc. Fla. State Hort. Soc. 113: 2000. Table 1. Embryo rescue and ploidy level of the offsprings from diploid-by-tetraploid crosses. Parents Number Ploidy Seed Pollen Fruit Seed Embr. Plant 2N +2N 3N 4N Apireno Valencia + Femminello *18 — — 72 62 0 8 2 De Hamlin + Femminello *10 — — 45 28 0 14 3 Continella Milam + Femminello 9 58 38 10 6 0 2 2 Key lime + Valencia 11 115 67 15 8 1 5 1 4X Femminello 3 4 3 3 2 1 0 0 Lisbon Valencia + Femminello 2 105 44 3 1 0 1 1 Hamlin + Femminello 1 21 21 28 26 2 0 0 Milam + Femminello 3 153 86 7 4 0 2 1 Key lime + Valencia 2 34 17 4 1 0 2 1 4X Femminello 3 76 59 26 26 0 0 0 Todo Del Valencia + Femminello 2 53 23 1 0 0 0 1 Hamlin + Femminello 2 11 8 2 1 0 1 0 Año Milam + Femminello 5 201 63 15 9 0 1 5 Key lime + Valencia 7 187 174 19 8 0 6 5 Bearss Hamlin + Femminello 1 1 1 0 0 0 0 0 Lemon Key lime + Valencia 4 50 21 4 1 0 3 0 Eureka 4X Femminello 5 80 123 100 99 0 1 0 *Fruit collected from two seasons, the other data correspond to one season. +Indicates a somatic hybrid. Table 2. Embryo rescue and ploidy level of the offsprings from allotetraploid by diploid-or-allotetraploid crosses. Parents Number Ploidy Seed Pollen Fruit Plant 2N +2N 3N 4N Hamlin + Femminello Key lime 2 9 0 0 3 6 Key lime + Valencia Key lime 5 17 0 0 4 13 Valencia + Femminello Key lime + Valencia 1 3 0 0 0 3 +Indicates a somatic hybrid. very low (1/129) among the total offsprings from crosses that in- hybrid rootstocks in preparation for field evaluation. Further re- cluded 4´ Femminello as pollen parent (Table 1). search is being conducted to improve germination and survival of The success of embryo rescue was notably affected by the de- triploid hybrid embryos, and to identify superior parental combina- velopmental embryo stage. Many embryos in globular and heart tions. Additional parents were utilized in the spring 2000 crosses, stages did not show any further development on EME medium, including ‘Lakeland’ limequat to potentially improve disease re- while embryos in more advanced development germinated suc- sistance in resulting triploid progeny. cessfully. Globular embryos from interspecific citrus crosses also failed to develop on a modified White’s medium supplemented Literature Cited with different addenda (Rangan et al., 1969). Conversely, early embryo stages from Citrus aurantium germinated at very high per- Arumuganathan, K. and E. D. Earle. 1991. Estimation of nuclear DNA content of centage on MS supplemented with malt extract (Carimi et al., plants by flow cytometry. Plant Mol. Biol. Rptr. 9:221-231. 1998). These contrasting results might be related with the triploid Carimi, F. F., De Pasquale and A. M. Puglia. 1998. In vitro rescue of zygotic em- bryos of sour orange, Citrus aurantium L., and their detection based on RFLP characteristic of the new hybrids in addition to sexual incompati- analysis. Plant Breeding 117:261-266. bility present between tree parents. Additionally, a few plants died Esen, A. and R. Soost. 1973. Seed development in citrus with special reference to when cultured on RMAN, and a few more during acclimatization. 2X x 4X crosses. Amer. J. Bot. 60:448-462. In general, the apical meristems of some seedlings stopped grow- Grosser, J. W. and F. G. Gmitter, Jr. 1990. Protoplast fusion and citrus improve- ing before or after the first true leaves, then died. ment. Plant Breeding Rev. 8:339-374. Grosser, J. W., G. A. Moore and F.
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