Acid Citrus Fruit Cultivar Improvement Via Interploid Hybridization

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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 medium 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 collected 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 embryos 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. G. Gmitter, Jr. 1989. Interspecific somatic hy- The occurrence of triploid hybrids was higher from tetraploid- brid plants from the fusion of ‘Key’ lime (Citrus aurantifolia) with ‘Valencia’ by-diploid crosses than their reciprocals (Tables 1 and 2), although sweet orange (Citrus sinensis) protoplasts. Scientia Hort. 39:23-29. the fruit set and the number of seeds per fruit were lower (Essen Rangan, T. S., T. Murashige and W. P. Bitters. 1969. In vitro studies of of zygotic and Soost, 1972). Even though the polyembryony present in so- and nucellar embryogenesis in Citrus. Proc. 1st Int. Citrus Symp. 1:225-229. Tusa, N., J. W. Grosser and F. G. Gmitter, Jr. 1990. Plant regeneration of ‘Valencia’ matic hybrids can hamper pollination success, this problem can be sweet orange, ‘Femminello’ lemon, and the interspecific somatic hybrid follow- overcome by increasing the amount of pollinations. ing protoplast fusion. J. Amer. Soc. Hort. Sci. 115:1043-1046. Embryo culture is a useful technique for citrus triploid production. Resulting triploids have been budded to precocious somatic

Proc. Fla. State Hort. Soc. 113: 2000. 125 Tusa, N., J. W. Grosser, F. G. Gmitter, Jr. and E. S. Louzada. 1992. Production of tetraploid somatic hybrid breeding parents for use in lemon cultivar improvement. HortScience 27:445-447.

Reprinted from Proc. Fla. State Hort. Soc. 113:126-130. 2000. FACTORS INVOLVED IN SOLUBLE SOLIDS ACCUMULATION IN CITRUS FRUITS

BRANDON R. HOCKEMA AND ED ECHEVERRÍA The mechanism involved is more than concentration by dehydra- University of Florida, IFAS tion as there is active accumulation of solids in fruit (Mills et al., Citrus Research and Education Center 1996; Yakushiji et al., 1996). Increasing fruit soluble solids during 700 Experiment Station Road drought involves one or more of the following processes, such as Lake Alfred, FL 33850 sugar movement, accumulation, and/or storage into fruit. Howev- er, the precise mechanisms are still unclear. Additional index words. Sink strength, citrus, sucrose synthase. The direct movement of assimilated carbon into a particular plant organ is determined by its ‘sink strength’ and by photosyn- Abstract. The direction of photosynthate movement among thesis in source tissue. ‘Sink strength’ is the ability of a particular plant organs is determined by “sink strength”, a model involv- organ to attract photoassimilates (Ho, 1988). During fruit elonga- ing the importation of sucrose (or other sugars) by hydrolysis tion and expansion, fixed carbon is required to provide growing tis- or sequestration. In most plants, sink strength is determined sues with energy for metabolism and to provide osmotic solutes to by the activities of two sucrose-cleaving enzymes: sucrose maintain turgor pressure. Fixed carbon is transported through the synthase(SS) and invertase(INV). In addition, activities of su- phloem in the form of sucrose, a disaccharide composed of joined crose phosphate synthase(SPS), sucrose phosphate phos- molecules of fructose and glucose. To develop a concentration gra- phatase(SPP), and tonoplast-bound ATPase could affect sink dient for adequate sink strength, cells must cleave sucrose, or ef- strength. Increased activities of membrane-bound sucrose fectively sequester it into the vacuole as in sugar beets (Getz et al., transporters or decreased vacuolar pH could also enhance accumulation of soluble solids. In fruits of many commercial 1991). Therefore, sink strength is determined by the ability of the crops, increases in soluble solids have been recorded during sink to metabolize sucrose and/or by its capacity for compartmen- drought conditions. This research investigates the determi- tation and storage. In plants, there are two specific enzymes capa- nants of citrus fruit sink strength through drought stress. Pot- ble of cleaving sucrose. The first is invertase (INV), whose ted Hamlin orange trees were grown under watered or unidirectional catalytic action yields fructose and glucose. The sec- drought-stressed conditions and fruit harvested and analyzed ond enzyme is sucrose synthase (SS) with a reversible reaction us- for Brix and acids. Stressed fruits had higher acid content and ing sucrose and UDP to yield UDP-glucose and fructose. soluble solids, and lower pH than controls. The following com- Sequestering sucrose in vacuoles permits the sink cell to maintain ponents of fruit sink strength were measured: SS, INV, SPS, a sucrose gradient between itself and the phloem, allowing the con- SPP, ATPase, PPase. In addition, isolated and purified mem- tinuous movement of sucrose toward the sink. branes from fruit were tested for the presence of a sucrose symport at the plasmalemma and an antiport at the tonoplast. Many storage organs appear to require the resynthesis and stor- Increased sink strength appeared to be the result of SS, since age of sucrose into the vacuole in a seemingly ‘futile’ cycle (Ho, SS activity was higher in drought-stressed versus well-wa- 1988). Related sucrose-metabolizing or synthesizing enzymes that tered fruit. Activities of other enzymes and transporters were may play a role in sink strength are sucrose phosphate synthase not significantly different between control and treated fruit. We (SPS, UDPG + F-6-P«S-6-P + UDP) and sucrose phosphate concluded that SS is the predominant factor controlling Brix phosphatase (SPP, S-6-P®Sucrose + Pi), although their exact levels in citrus fruit, although the altered pH could have con- functions in a sink organ are uncertain. Other factors such as low tributed to sink strength by enhancing acid hydrolysis. vacuolar pH may be involved in sink strength, since low pH can cleave sucrose (Wienen and Shallenberger, 1988). In citrus, the It is well known that some temperate fruits accumulate higher vacuoles of fruit juice cells can be extremely acidic with a pH of 3 levels of soluble solids during mild drought stress (Behboudian and or lower (Echeverria and Burns, 1989). Such low pH is capable of Mills, 1997). Since fruit quality and production is not compro- hydrolyzing sucrose into glucose and fructose in vitro (Wienen and mised by mild late-season drought but rather enhanced, this issue Shallenberger, 1988). That same low pH could automatically has attracted the interest of many researchers and fruit producers. cleave sucrose entering the vacuole at a rate dependent on the hy- dronium ion concentration and temperature. A fruit cell could use Florida Agricultural Experiment Station Journal Series No. N-01941. We are a sucrose antiport at the tonoplast to sequester sucrose into the vac- grateful for the assistance of Dr. Larry Parsons in using his equipment in measuring uole, utilizing the existing ýpH (Getz et al., 1991). water potential, and to Pedro Gonzalez for assistance.

126 Proc. Fla. State Hort. Soc. 113: 2000.