HORTSCIENCE 25(4):474-477. 1990.

t

u

d

A

branched. Callus formation was particularlypronounced on explants cultured on medium

Regeneration and Fof ‘Burpless HybridCultured from ExciW. Msikita1, R.M. Skirvin2, J.A. JN. Ali1

Department of Horticulture, Universi

Additional index words. cotyledons, growth

Abstract. Seeds of ‘Burpless Hybrid’ cucfrom their seedcoats and excised into pieccotyledons. The seed pieces were disinfesteSkoog medium with 16 combinations of B(0.0,0.1,0.2,0.3 mg·liter-1). On all media, whole plants within 4 weeks, but did notshoots and male flowers on most media. shoot regeneration and the most female flowBAP and 0.3 mg NAA/liter. One of the fedeveloped a small fruit with viable seed.

In vitro regeneration of cucumbers has beena subject of considerable interest, and var-ious explants have been used to develop calliand regenerate plants. Explants that have re-generated shoots have included seedling cot-yledons (Kim and Jang, 1984; Kim et al.,1988; Sekioka and Tanaka, 1981), hypocot-yl segments (Rajasekaran et al., 1983), leafblades and petioles (Wehner and Loey, 1981),

and axillary buds (Handley and Chambliss,1979).

A tissue culture system that would enablesuccessful regeneration from mature, ex-cised dry seed pieces would offer a meansto rapidly multiply plants when only a lim-ited number of seed is available. Cade et al.(1987) reported shoot regeneration fromcotyledons of Cucumis sativus L. after sub-culturing and/or transferring to another typeof medium and maintaining the explants inthe dark. Lange and Juvik (1986) also re-ported regeneration from mature seed coty-ledons of several Cucurbita species. Msikitaet al. (1988) recently reported preliminaryobservations regarding shoot regenerationfrom several cucumber cultivars and lines,and observed that cucumber shoot regener-ation and subsequent flowering varied amonggenotypes and type of explant. In this paper,we report shoot regeneration and floweringof the cucumber cultivar Burpless Hybrid at

Received for publication 9 Dec. 1988. This re-search was supported in part by the Univ. of Il-linois Agricultural Experiment Station (Hatch 65-0323), the Swedish International DevelopmentAuthority (SIDA), and the Zambian Ministry ofAgriculture and Water Development (MAWD). Wethank Hassan Abu-Qaoud for the statistical analy-sis. The cost of publishing this paper was defrayedin part by the payment of page charges. Underpostal regulations, this paper therefore must behereby marked advertisement solely to indicate thisfact.1Graduate Student.2Professor.3Associate Professor.

474

with 2 mg BAP and 0.2 mg NAA/liter. Someof this callus persisted until the plants wereremoved to soil.

Some cultures developed 2 to 3 axillaryshoots. Within 3 to 4 weeks of culturing, theshoots were large enough to transplant to soil.No flowering was observed in vitro.

Cotyledons: shoot regeneration. Within 2weeks, cotyledons expanded to more than 10times their original size (Fig. 1, top). By the3rd week of culturing, callus formed in theregion of the cut surface. Some callus rap-idly developed organized structures (Fig. 1,top). Some of these structures became plant-lets within 6 to 8 weeks of explanting. Some

lowering in Vitro’ Cucumbersed Seeduvik3, W.E. Splittstoesser2, and

y of Illinois, Urbana, IL 61801

regulators, Cucumis, tissue culture

mber (Cucumis sativus L.) were separatedes comprising an embryonic axis and two and explanted on modified Murashige andAP (2.0, 3.0, 4.0, 5.0 mg·liter -1) and NAAembryonic axes germinated and grew intoflower. Cotyledons developed adventitious few female flowers developed. The best

ers were observed on medium with 2.0 mgmale flowers was pollinated in vitro and it

various concentrations of BAP and NAA.Cotyledons and embryonic axis explants.

Seed of ‘Burpless Hybrid’ cucumber (pro-vided by Sakata Seed Co., Japan) were sep-arated from their seedcoats and cut into piecesconsisting of an embryonic axis and twocotyledons. The pieces were disinfested (10min) in 10% (v/v) Clorox, to which 40 mgof Alconox powder had been added as a sur-factant, and then rinsed five times (30 see/rinse) with sterile water. The pieces from asingle seed were maintained as a clone andaseptically transferred to 25 × 150-mm cul-ture tubes (one seed piece per tube) contain-ing 10 ml of modified Murashige and Skoog(MS) (1962) high salt medium supplementedwith 16 combinations of BAP and NAA (Ta-ble 1). Before autoclaving (1.06 kg·cm-2

pressure, 15 min), 10 ml of Staba (1969)vitamins were added. The pH of the mediawas adjusted to 5.7, and 7% (w/v) DifcoBacto-agar was added. Explants were placedinto the media vertically, with the basal cutend ≈1 to 2 mm beneath the surface of the

medium.

The experimental treatment consisted of25 seeds divided into two cotyledons and oneembryonic axis each (75 explants × 16 me-dia). Cotyledon explants were grown for 8weeks under 16-hr daylength in a cultureroom. Light (40 µmol·s-1·m -2) was pro-vided by cool-white fluorescent lamps. Thetemperature was maintained at 25 ± 2C.The experiment was repeated three times andwas designed to be a 4 × 4 factorial, with25 pairs of cotyledons as replications for fourlevels each of BAP and NAA. Data wereanalyzed using the General Linear Modelprocedure of SAS (1982). The cultures wereexamined weekly for their growth, shoot re-generation, and flowering.

To assess the type of regeneration, someof the regenerating cotyledons were embed-ded in paraffin and later 10-µ pieces weresectioned on a microtome, mounted on glassslides, and stained in safranin and fast green

as described by Johansen (1940).Transfer of in vitro-derived plants to soil.

Some of the shoots obtained in this studywere transplanted to small plastic pots (0. 15liters) containing a 1 sterilized peat :1 perlite: 1 soil mixture (by volume) and placed inthe culture room under the conditions de-scribed earlier. After 5 to 7 days, plantletswere transferred to a greenhouse.

In vitro pollination. At anthesis, some ofthe female flowers that developed in vitrowere pollinated from male, greenhouse-growncucumber flowers. To reduce potential con-tamination of the tissue culture medium dur-ing pollination, the greenhouse-grown maleflowers were bagged 1 day before anthesis.On the following day, the flowers were re-moved and pollen was carefully extracted ontoa sterile petri dish with a sterile scapel. Usinga small brush (sterilized by autoclaving),pollen was carefully transferred onto thestigmas of two female cucumber flowers invitro.

To verify pollen viability, pollen fromgreenhouse- and in vitro-derived male flowerswas stained with acetocarmine and examinedunder a microscope.

After 3 days of culturing, explants beganto expand and turn green. Subsequent growthwas different for each explant type.

Embryonic axes. The embryonic axes ger-minated, a radicle grew from the axis, andit thickened with callus. Callused roots later

plantlets produced flowers (Fig. 1, bottom).Shoots arose adventitiously from the callus(Fig. 2). Shoots had no preformed root sys-tem, suggesting they arose via organogene-sis.

HORTSCIENCE , VOL. 25(4), APRIL 1990

HORTSCIENCE , VOL. 25(4), APRIL 1990

Shoots were obtained at each level of BAPand NAA. There was no significant inter-

action between NAA and BAP for shoot re-generation. The best shoot regeneration wason medium supplemented with 2 mg BAP/liter. Shoot regeneration was significantlyreduced by increasing BAP concentration from2 to 5 mg·liter-l (Fig. 3). The highest av-erage shoot regeneration due to NAA wasobserved between 0.2 and 0.3 mg·liter-1 (Fig.4); beyond 0.3 mg·liter-1

, average shootnumber declined.

Flowering. By the 7th week of culture,some of the shoots developing on cotyledonsbegan to flower. Male flowers (Fig. 1, bot-tom) were observed on all media, except thatsupplemented with 5 mg BAP/liter alone. Themost male flowers were produced at 4 mgBAP/liter with various combinations of NAA(Table 1). Female flowers were observed onlyon media supplemented with either 2 mg BAPand 0.3 mg NAA/liter or 4 mg BAP/literalone. On the latter medium, female flowerswere very poorly developed.

475

In vitro pollination. Each of the femaleflowers pollinated in vitro developed a smallcucumber fruit (Fig. 1, far right) that wereharvested from the culture tube at maturity,i.e., when they turned yellow. One of the

476

fruits contained two seeds. One of the seedswas fully developed, while the other wasempty. The second fruit contained no seed.The embryo was removed from the viableseed and recultured; it germinated after 5

days and grew into a plant.The viability of the pollen from in vitro-

derived male flowers was verified by stain-ing with acetocarmine. Most of the pollengrains ( >95%) stained red, were round, andsimilar in appearance to those from a green-house plant.

In this study, the best shoot regeneration(50%) from cotyledons was obtained on me-dia supplemented with either 2 mg BAP and0.3 mg NAA/liter or 4 mg BAP and 0.3 mgNAA/liter. Cade et al. (1987) reported re-generation from cucumber seed cotyledonscultured on MS media supplemented withvarious combinations of BAP, NAA,and 2,4-D and kept in the dark for 4 to 6weeks; however, they did not indicate re-generation frequency or flowering. In ourstudy, shoots were regenerated directly fromdried seed cotyledons in 6 to 8 weeks.

The consistent formation of organizedstructures proximal to the cut surface of iso-lated cotyledons was also noted in our earlierreport (Msikita et al., 1988). The polarity ofshoot regeneration suggests a gradient ofgrowth-promoting factors within the cotyle-dons and/or the translocation of growth-p ro-moting substances toward the embryonic axis.

The absence of flowers on embryonic axes,in vitro suggests the stimulus for flower for-mation on preformed embryonic axes is dif-ferent from that of adventitious shoots. Thepresence of the embryonic axis (a preformedshoot) also may impart juvenility to the re-generated shoot (Scorza, 1982). Rajasekaranet al. (1983) observed both male and femaleflower formation in vitro on cultured hypo-cotyl segments of ‘Superpickle’ cucumber.Flowers were observed on hypocotyl-derivedshoots after 20 weeks on MS medium supple-mented with BA (0.5 or 1.0 µM) and 2,4-D(1.5 or 5.0 µM). The ability of these flowersto set seed or serve as a pollen source wasnot reported. In our study, the ability of thefemale flowers to set fruit and viable seed,as well as the viability of the pollen from invitro-derived male flowers, was demon-strated.

The protocol developed in this study of-fers an in vitro method to regenerate cucum-

bers from dried seed cotyledons andembryonic axes. In addition, this system maybe useful to 1) obtain many plantlets from asingle seed, 2) study the control of floweringin vitro, and 3) serve as a possible alternativeto conventional methods of cucumber breed-ing, e.g., the production of somaclonal vari-ants (Larkin and Scowcroft, 1981).

Literature CitedBottino, P.J. 1981. Vegetable crops, p. 141-164.

In: B.V. Conger (ed.). Cloning agricultural plantsvia in vitro techniques. CRC Press. Boca Ra-ton, Fla.

Cade, R.M., T.C. Wehner, and F.A. Blazich.1987. Organogenesis and embryogenesis fromcucumber (Cucumis sativus L.) cotyledon-de-rived callus. HortScience 22:1130. (Abstr.)

Garcia-Sogo, M., I. Granelli, and B. Garcia-Sogo.1986. Plant regeneration from explant-derivedcalli of Cucumis anguria L. var. longipes. Cu-curb. Genet. Coop. 9:108-109.

HORTSCIENCE, VOL. 25(4), APRIL 1990

Handley, L.W. and O.L. Chambliss. 1979. In vi-tro propagation of Cucumis sativus L. Hort-Science 14:22-23.

Johansen, D.A. 1940. Plant microtechnique, p.145–154. McGraw-Hill, New York.

Kim, S.G., J.R. Chang, H.C. Cha, and K.W.Lee. 1988. Callus growth and plant regenera-tion in diverse cultivars of cucumber (Cucumissativus L.). Plant Cell Tissue & Organ Cult.12:67-74.

Kim, S.G. and J.R. Jang. 1984. Regeneration ofplants from callus tissue of cucumber (Cucumissativus L.) seedling cotyledons. Plant Physiol.75:15. (Abstr.)

Lange, N.E. and J.A. Juvik. 1986. Organogenesisfrom explants of mature seed cotyledons of 20accessions from the genera Cucurbita, Cucumisand Citrullus. HortScience 21:687. (Abstr.)

HORTSCIENCE , VOL. 25(4), APRIL 1990

Larkin, P.J. and W.R. Scowcroft. 1981. Soma-clonal variation—A new source of variation fromcell cultures for plant improvement. Theor. Ap-plied Genet. 60:197-214.

Msikita, W., R.M. Skirvin, J.A. Juvik, and W.E.Splittstoesser. 1988. In vitro regeneration andflowering of cucumber cultivars and lines cul-tured from excised seed. Cucurb. Genet. Coop.11:5-7.

Murashige, T. and F. Skoog. 1962. A revised me-dium for rapid growth and bioassays with to-bacco tissue cultures. Physiol. Plant. 15:474-497.

Rajasekaran, K., M.G. Mullins, and Y. Nair. 1983.Flower formation in vitro by hypocotyl explantsof cucumber (Cucumis sativus L.). Ann. Bot.52:417-420.

SAS Institute, Inc. 1982. SAS user’s guide. SASInst., Cary, N,C.

Scorza, R. 1982. In vitro flowering. p. 106-127.In: J. Janick (ed.). Horticultural reviews. vol.4. AVI Publishing Co., Westport, Conn.

Sekioka, A.J. and J.S. Tanaka. 1981. Differen-tiation in callus cultures of cucumber (Cucumissativus L.). HortScience 16:451. (Abstr.)

Staba, J.E. 1969. Plant tissue culture as a tech-nique for the phytochemist, p. 75–106. In: M.K.Seikel and V.C. Runeckles (eds.). Recent ad-vances in photochemistry. vol. 2. Appleton-Century Crofts, N.Y.

Wehner, T.C. and R.D. Loey. 1981. In vitro ad-ventitious shoot and root formation of cultivarsand lines of Cucumis sativus L. HortScience16:759-760.

477