m

o r1

FEATURE

The Use of E

Horticultural Crops Research Lab202

BackgroundThe first successful systematic culture of

plant embryos on an artificial medium of de-fined chemical composition was by E. Han-ning in 1904 with mature embryos of twocrucifers (Narayanaswami and Norstog,1964). Then, in 1929, Laibach (Mahesh-wari, 1966) pointed out the potential appli-cation of embryo culture in plant breeding torescue embryos from interspecific hybrids thatotherwise fail due to embryo abortion. Tu-key’s (1933b) culture of cherry embryos in1933 was a milestone in embryo culture offruit crops. His medium and procedure has

been widely used and even applied to othercrops, such as cucurbits (Whitaker and Davis,1962). Using this procedure, Blake (1939)was the first to employ culture in a peachbreeding program.

LaRue (Narayanswami and Norstog, 1964)successfully cultured and grew small (0.5-mm-long) embryos of many genera as earlyas 1936 with the addition of inorganic salts,sugar, yeast extract, and indolebutyric acidto the medium. However, embryos in theglobular stage and smaller are very difficultto culture successfully. Maheshwari (1958)demonstrated by use of in ovule embryo cul-ture that it is possible to grow a four-celledproembryo of Papaver somniferum L. to ma-turity. The successful growth of ovule-cul-tured embryos depends on their stage ofmaturity at the time of culture, genotype,culture medium, and culture environment.

The developing embryo is a dynamic sys-tem that has changing requirements as it ma-

Received for publication 8 Mar. 1989 The costof publishing this paper was defrayed in part bythe payment of page charges. Under postal regu-lations, this paper therefore must be hereby markedadvertisement solely to indicate this fact.

HORTSCIENCE , VOL. 25(4), APRIL 1990

bryo Culture in FDavid W. Ramming

ratory, U.S. Department of Agricultu South Peach Avenue, Fresno, CA 93

tures. The smaller the embryo, the morecomplex the medium required. Raghavan(1976) states in his review of embryo culturethat there are heterotropic and autotropicstages in embryo development. The embryoin the heterotropic stage of development issmaller than in the autotrophic stage andusually requires the presence of growth reg-ulators to allow for proper development. Theautotropic stage begins at about the late heartstage and development of such an embryodoes not depend on exogenous sources ofgrowth regulators, thus making it more ame-nable to in vitro culture.

Embryo culture is useful when there is poorembryo development or abortion. Embryoabortion occurs in early ripening genotypesof Prunus where the flesh matures beforeseed maturity (Tukey, 1933a; Tukey and Lee,1937), precluding their use as females in abreeding program. The poor germination of

seeds from early ripening grapes is probablydue to poor embryo development. Embryoshave been rescued from early ripening Pru-nus (Tukey, 1934) and Vitis (Ramming andEmershad, 1984) cultivars, thus allowing theiruse as females. Many seedless grape culti-vars are stenospermic (Stout, 1936), i.e., theyare characterized by cessation of embryo de-velopment after fertilization and failure ofseed development. Embryos have been res-cued and grown into plants from stenosper-mocarpic seedless grapes (Emershad andRamming, 1984; Spiegel-Roy et al., 1985),allowing growth of progeny resulting fromthe hybridization of seedless with seedlessgrapes. The production of interspecific hy-brids is useful for the transfer of desirablegenes from wild to cultivated species. In manycases, wide crosses between species are dif-ficult to produce because of many factorsthat act as barriers. Postzygotic barriers suchas endosperm abortion are a common occur-

ruit Breeding

e, Agricultural Research Service,727

rence, but they have been overcome throughthe use of embryo rescue and many inter-specific hybrids have been produced (Collinsand Grosser, 1984). Embryo rescue has alsobeen used to overcome postzygotic barriersduring self- and cross-incompatibility (Fas-suliotus, 1977; Cox et al., 1960; Fridrikssonand Bolton, 1963).

Embryo culture has been used for rapidseed germination tests of peach (Tukey, 1944;Flemion, 1936) and other woody plants (Fle-mion, 1941, 1948); for production of virus-free citrus plants through culture of nucellarembryos (Bitters et al., 1970, 1972); and forstudying the developmental and growth re-quirements of embryos (Steward and Hsu,1977). Embryo culture has also been used toshorten breeding cycles in apple (Nickell,1951) and iris (Randolph, 1945), where ef-fective germination was reduced from yearsto months by overcoming dormancy. The

possibility also exists for using embryo cul-ture as a screening procedure. Landa et al.in 1980 (Collins and Grosser, 1984) inves-tigated the possibility of selecting mutantswith high amino acid content. Pollen mightalso be screened for temperature tolerance(Zamir and Vallejos, 1983) before in vitrofertilization of ovules. Embryo culture hasbeen reviewed by Sanders and Ziebur (1963),Narayanaswami and Norstog (1964), Ma-heshwari (1966), Raghavan (1966, 1976,1977, 1980), Bhojwani and Razdan (1983),and Collins and Grosser (1984). One of thefew reviews of ovule culture was by Rangan(1984). This review will cover the use ofembryo rescue for two fruit crops, Prunusand Vitis.

Results achievedPeach embryo culture. After Tukey suc-

cessfully cultured cherry embryos (1933 b),

393

others applied it in their breeding programs.Blake (1939) used the procedure and Houghimprove on the medium and procedure (Smithet al., 1969). Embryos with 75% fill (PF1)could be rescued with this procedure. Peachembryo culture was started in 1975 at theHorticultural Crops Research Laboratory,Fresno, Calif., using Smith, Bailey, andHough (SBH) medium (Smith et al., 1969)and procedures to develop progeny from earlyripening genotypes and increase the chancesof developing good early ripening cultivars.The technique of Smith et al. (1969) of cub-ing the fruit to remove all skin, surface-ster-ilizing with phenol, cracking the pit, surface-sterilizing the seed with merthiolate, andrinsing three times with distilled water wasvery time consuming and could be toxic topeople. Since techniques and procedures usedin breeding programs must be efficient, sim-ple, and able to process many embryos, the

394

above procedure was simplified. Our pro-cedure, in Fresno, for embryo culture con-sisted of cracking the fruit and pit open toremove the seed and then flaming the seedin the laminar flow hood before extractingthe embryo and placing it into the culturemedium. Care must be taken to prevent dam-age during flaming. Damage occurs to theintegument during flaming and the uptake ofnutrients is reduced during ovule culture ofvery small embryos; hence, an alternativesterilization procedure for ovule culture wasdeveloped. The fruit is surface-sterilized with70% ethanol for 30 sec, then with 0.525%Na hypochlorite for 5 min before it is cutopen under sterile conditions and the seed isplaced directly in the culture medium. Caremust be exercised so the seed is not damagedor taken from open pits that generally arecontaminated. Embryos from soft fruit havea greater chance of becoming contaminated

because of free-flowing juice.The first population; from six early rip-

ening genotypes, having 70- to 72-day fruitdevelopment periods, were developed usingSBH medium. Without embryo culture, plantscould not be developed from these geno-types. The procedure allowed the selectionof genotypes ripening 10 to 30 days earlierthan the parents from these populations (Ta-ble 1). Embryo development in these veryearly ripening selections was less than in theparents; hence, the SBH medium was inad-equate to support their growth. A more-de-fined medium was developed for the

successful culture of these genotypes. Mu-rashige and Skoog (MS) (1962) medium withthe addition of 1240 mg potassium succinateand 584.6 mg L-glutamine per liter and 3%sucrose allowed embryos as small as 5 mmlong (PF1 = 25) to be grown successfully.This was first used in the breeding programin 1980. All embryos longer than 10 mm areroutinely cultured on SBH and those 5 to 10.mm long on MS. The recovery of viable plantsfrom ovules and embryos has ranged from41% to 79% since 1976. Contamination isonly a problem if inexperienced and un-trained people perform the embryo culture.The successful use of embryo culture in ourpeach and nectarine breeding program from1975 to 1987 is shown in Table 2. Embryos<10 mm long are more difficult to growsuccessfully even though they are grown ona more complex medium (Table 3). Only32% of such embryos produced plants, com-

HORTSCIENCE , VOL. 25(4), APRIL 1990

pared to 78% for larger embryos.Some recent selections ripen so early that

their embryos are only 1 mm long at fruitmaturity. The use of these selections as fe-males required improvements in media andprocedures. The cotton ovule culture me-dium of Stewart (1979) was tested for Pru-nus (Ramming, 1985), and it provided thenecessary environment for the developmentof embryos initially 1.0 to 5.0 mm long.Cotyledons are well defined at this stage,and sucrose concentration was found to bemore critical to embryo development andsubsequent growth than cytokinins, auxins,

HORTSCIENCE , VOL. 25(4), APRIL 1990

or additional organic addenda. Sucrose at 6%to 8% during ovule culture was optimum forembryo fresh and dry weight accumulationand germination (Figs. 1 and 2). Precociousgermination is prevented during ovule cul-ture by the integuments, allowing the em-bryos to increase in fresh and dry weightwithin the integuments. At this time, the em-bryo is using the nucellus and endosperm aswell as the nutrient medium for growth. Em-bryos were excised after 2 weeks of ovuleculture and sub-cultured at various sucroselevels during stratification at 0.5C. Embryodevelopment continued during stratification,

increasing in fresh weight by 35% to 129%(Fig. 1). Lower sucrose levels (0% and 2%)at this time were found to be the most ben-eficial for increase in fresh and dry weightand germination (Figs. 1 and 2). When ovuleculture was first used in 1981, the embryoswere not excised or subculture, but treatedlike mature seeds during stratification andgermination. A higher percentage of plantsdeveloped when the embryos were excisedafter the initial 2 weeks of ovule culture andthen subculture on either MS or SBH dur-ing the stratification period than when theywere treated like mature seed. This superiorprocedure has been used since 1983.

We have made many selections fromseedlings produced by embryo culture meth-— —ods. Two cultivars —Goldcrest peach andMayfire nectarine—have resulted from em-bryo culture and are among the earliest ofthe commercially grown cultivars in Cali-fornia (Ramming and Tanner, 1987a, 1987b).

The culture of zygotes could be useful forrescuing embryos from interspecific hybridsthat abort due to postzygotic barriers and forzygotic screening techniques. In 1986, weinduced embryos 7 to 10 days post-bloom toenlarge to ≈12 mm (300 mg). However, theembryos appeared to abort during stratifi-

cation, and no viable plants were produced.Hormones during the initial ovule culture pe-riod were necessary as for tomato, and levelsused were those described by Neal and To-poleski (1985). Additional work on Prunusis needed to be able to rescue zygotes.

Grape embryo culture. The seeds of manyearly ripening grapes do not germinate whenthey are stratified and planted in soil. Thesegenotypes are valuable as females and asmales to increase the chances of developingearly ripening grape cultivars. Therefore,peach embryo culture techniques were ap-plied to grapes. We used modified White’smacroelements medium with Norstog’s mi-croelements and vitamins plus glycine andcasein hydrolysate (Emershad and Ram-ming, 1984). Using this technique, we haveimproved initial germination from 0%-16%to 19%-24% and total plant production to30%-32% (Table 4). Seeds are cultured in-dividually in 20 × 150-mm test tubes, strat-

395

396

ified for 3 months at 0.5C, and then allowedto germinate. Some seed germinate imme-diately and are planted directly in the green-house. Some small embryos come out of theseed and germinate directly on the mediumand can be planted in the greenhouse whenlarge enough. Embryos from the ungermi-nated seed will grow when the seed is cutopen and the embryo extracted and sub-cul-tured. Even floaters (seed without endo-sperm) occasionally contain embryos that canbe successfully rescued, although not effi-ciently enough for a breeding program (Ta-ble 5). Results of culturing crosses from earlyripening seeded grapes for the breeding pro-gram as described above are shown in Table6.

Every grape breeder’s dream has been tointerbreed stenospermic seedless grapes. Thisstep holds the promise of eliminating the in-termediate seeded female and the secondgeneration for intermating or back crossing(Fig. 3). In addition to saving one generationof seedlings and ≈5 years; land, labor, fer-tilizer, and water are also saved. The geno-types can also be hybridized directly withoutbeing diluted by genes from a seeded female,making it easier to combine complementarytraits. The proportion of seedless progenyshould also be higher, requiring smaller pop-ulations to develop the same number of seed-less seedlings as produced in seeded × seedlesscrosses.

Plants from seedless genotypes were firstproduced by embryo culture in our labora-tory in 1981 (Emershad and Ramming, 1982).

Since the embryos ranged from four to 50cells at the time of culture, they were im-practical to culture directly as embryos. Theovule is cultured, allowing the embryo toenlarge directly in the integument, after whichthe embryo is excised and subculture, al-lowing it to germinate and grow into a plant.In 1983, ovules from 29 seedless genotypeswith varying sizes of seed traces were cul-tured from open-pollinated fruit (Table 7).There exists a range of responses to culture,as measured by the percentage of ovules withembryos, which is not correlated to seed traceweight (correlation coefficient = 0.19).During culture, multiple embryos develop insome ovules, usually as a result of somaticembryogenesis from the zygotic embryo. Thisconclusion was reached based on position ofmultiple embryos attached to zygote and onfruiting seedlings. The amount of multipleembryogenesis seems to be genotypic-de-pendent, based on data from culturing the

HORTSCIENCE , VOL. 25(4), APRIL 1990

HORTSCIENCE , VOL. 25(4), APRIL 1990

same genotypes for several years. Using ovule

culture, many progeny have been developedfrom seedless × seedless grape crosses (Ta-ble 8). The first family that fruited has shown82% seedlessness (Fig. 4) instead of the 15%average (Loomis and Weinberger, 1979)found in seeded × seedless progeny (Fig. 4).Genotypes with aborted seeds that are so smallthey are barely visible with the naked eye(<1 mm long) were found in 23% of theseedlings. One such seedling had larger ber-ries than either parent, which is very en-couraging in the development of naturallylarge fruit in seedless grapes.

Embryo culture can be a useful tool inbreeding fruit crops and we have exploredonly a few of these. In addition to being ableto culture embryos before abortion, it wouldbe extremely valuable to be able to performin vitro pollination and fertilization. Cross-ing barriers could be eliminated and the po-tential to screen male and female gametesbefore hybridization might be possible.

Literature Cited

Bhojwani, S.S. and M.K. Razdan. 1983. Plant

tissue culture: Theory and practice. Elsevier,Amsterdam. p. 199-236.

Bitters, W.P., T. Murashige, T.S. Rangan, andE. Nauer. 1970. Investigations on establishedvirus-free plants through tissue culture. Calif.Citrus Nurserymen’s Soc. 9:27-30.

Bitters, W. P., T. Murashige, T.S. Rangan, andE. Nauer. 1972. Investigations on establishedvirus-free plants through tissue culture, p. 267–271. In: W.C. Price (ed.). Proc. 5th Conf. Intl.Organization Citrus Virologist. Gainesville, Fla.

Blake, M.A. 1939. Some results of crosses of earlyripening varieties of peaches. Proc. Amer. Soc.Hort. Sci. 37:232-241.

Collins, G.B. and J.W. Grosser. 1984. Culture ofembryos, p. 241–257, In: I.K. Vasil (ed.). Cellculture and somatic cell genetics of plants. vol.1. Laboratory procedures and their applications.Academic, Orlando, Fla.

Cox, E.A., G. Stotzky, and R.D. Goors. 1960.In vitro culture of Musa balbisiana Colla em-bryos. Nature (London) 185:403-404.

Emershad, R.L. and D.W. Ramming. 1982. In-

397

Tukey, H.B. 1933a. Embryo abortion in early-

OVUlO embryo culture of Vitis vinifera L. C.V.‘Thompson Seedless’. HortScience 17(4):576.(Abstr.)

Emershad, R.L. and D.W. Ramming. 1984. In-OVU1O embryo culture of Vitis vinifera L. C.V.‘Thompson Seedless’. Amer. J. Bot. 71:873-877.

Fassuliotus, G. 1977. Self fertilization of Cucumismetuliferus Naud. and its cross compatibilitywith Cucumis melo L. J. Amer. Soc. Hort. Sci.102:336-339.

Flemion, F. 1936. A rapid method for determiningthe germinative power of peach seeds. Contrib.Boyce Thompson Inst. 8:289-293.

Flemion, F. 1941. Further studies on the rapiddetermination of the germinative capacity of

peach seeds. Contrib. Boyce Thompson Inst.11:455-464.

Flemion, F. 1948. Reliability of the excised em-bryo method as a rapid test for determining thegerminative capacity of dormant seeds. Con-trib. Boyce Thompson Inst. 15:229-242.

Fridriksson, S. and J.L. Bolton. 1963. Prelimi-nary report on the culture of alfalfa embryos.Can. J. Bot. 41:439-440.

Loomis, N.H. and J.H. Weinberger. 1970. Inher-itance studies of heedlessness in grapes. J. Amer.Soc. Hort. Sci. 104:181-184.

Maheshwari, N. 1958. In vitro culture of excisedovules of Papaver somniferum. Science 127:342.

Maheshwari, P. 1966. The embryology of an-giosperms-A retrospect and prospect, p. 97-112. In: E. Cutter (ed.). Trends in plant mor-phogenesis. Wiley, New York.

Murashige, T. and F. Skoog. 1962. A revised me-dium for rapid growth and bioassays with to-bacco tissue cultures. Physiol Plant. 15:473-497.

Narayanaswami, S. and K. Norstog. 1964. Plantembryo culture. Bot. Rev. 30:587-628.

Neal, C-.A. and L.D. Topoleski. 1985. Hormonalregulation of growth and development of to-

398

mato embryos in vitro. J. Amer. Soc. Hort. Sci.110:869-873.

Nickell, L.G. 1951. Embryo culture of weepingcrabapple. Proc. Amer. Soc. Hort. Sci. 57:401-4 0 5 .

Raghavan, V. 1966. Nutrition, growth and mor-phogenesis of plant embryos. Biol. Rev. 41:1-58.

Raghavan, V. 1976. Experimental embryogenesisin vascular plants: Academic, London.

Raghavan, V. 1977. Applied aspects of embryoculture, p. 375–397. In: J. Reinert and Y.P.S.Bajaj (eds.). Applied and fundamental aspectsof plant cell, tissue, and organ culture.Springer-Verlag, New York.

Raghavan, V. 1980. Embryo culture. Intl. Rev.Cytol. Suppl. II. p. 209-240.

Ramming, D.W. 1985. In ovulo embryo culture

of early-ripening Prunus. HortScience 20:419–420.

Ramming, D.W. and R.L. Emershad. 1984. Em-bryo culture of early-ripening seeded grapegenotypes. HortScience 19:594. (Abstr.)

Ramming, D.W. and O. Tanner. 1987a. ‘Gold-crest’ peach. Fruit Var. J. 41:52–53.

Ramming, D. W. and O. Tanner. 1987b. ‘May-fire’ nectarine. Fruit Var. J. 41:80-81.

Randolph, L.F. 1945. Embryo culture of Iris seed.Bul. Amer. Iris Soc. 97:33-45.

Rangan, T.S. 1984. Culture of ovules, p. 227-231. In: I.K. Vasil (ed.). Cell culture and so-matic cell genetics of plants. vol. 1, Laboratoryprocedures and their applications. Academic,Orlando, Fla.

Sanders, M.E. and N.K. Ziebur. 1963. Artificialculture of embryos, p. 297–326. In: P. Ma-heshwari (ed.). Recent advances in the em-bryology of angiosperms. Intl. Soc. PlantMorphologists, Delhi, India.

Smith, C.A., C.H. Bailey, and L.F. Hough. 1969.Methods for germinating seeds of some fruitspecies with special reference to growing seed-

lings from immature embryos. New Jersey Agr.Expt. Sta. Bul 823.

Spiegel-Roy, P., N. Sahar, J. Baron, and U. Lavi.1985. In vitro culture and plant formation fromgrape cultivars with abortive ovules and seeds.J. Amer. Soc. Hort. Sci. 110:109-112.

Stewart, J. McD. 1979. Use of ovule cultures toobtain interspecific hybrids of Gossypium, p.45–56. In: J.T. Barber (ed.). Plant tissue cul-ture—Proceedings of a symposium. SouthernSection of Amer. Soc. of Plant Physiol. NewOrleans, La.

Stewart, J. McD. and C.L. Hsu. 1977. In-ovuloembryo culture and seedling development ofcotton (Gossypium hirsutum L.). Planta 137:113-117.

Stout, A.B. 1936. Seedlessness. in grapes. NewYork Agr. Expt. Sta. Tech. Bul. 238.

ripening varieties of Prunus avium. Bot. Gaz.94:433-468.

Tukey, H.B. 1933b. Artificial culture of sweetcherry embryos. J. Hered. 24:7–12.

Tukey, H.B. 1934. Artificial culture methods forisolated embryos of deciduous fruits. Proc. Amer.Soc. Hort. Sci. 32:313-322.

Tukey, H.B. 1944. The excised-embryo methodof testing the terminability of fruit seed withparticular reference to peach seed. Proc. Amer.Soc. Hort. Sci. 45:211-219.

Tukey, H.B. and F.A. Lee. 1937. Embryo abor-tion in the peach in relation to chemical com-position and season of fruit ripening. Bot. Gaz.98:586-597.

Whitaker. T.W. and G.N. Davis. 1962. Cucurbitsbotany; cultivation, and utilization. Leonard Hill,London.

Zamir, D. and E.C. Vallejos. 1983. Temperatureeffects on haploid selection of tomato micro-spore and pollen grains, p. 335-342. In: D.L.Mulcahy and E. Ottaviano (eds.). Pollen: Bi-ology and implications for plant breeding, El-sevier, Amsterdam.

HORTSCIENCE , VOL. 25(4), APRIL 1990