Cucurbit Genetics Cooperative Report 13:14-17 (Article 6) 1990

Effect of Explant Age and Growth Regulator Concentration on Adventitious Shoot Formation from Cucumber Cotyledonary Tissue

Rebecca M. Cade, Todd C. Wehner and Frank A. Blazich

Department of Horticultural Science, Box 7609, North Carolina State University, Raleigh, NC 27695-7609

(This paper is based on a portion of a thesis that was submitted by the senior author in partial fulfillment of the requirements for the M.S. degree.)

Much of the work on organogenesis of cucumber (Cucumis sativus L.) has been difficult to repeat, and results have often been unpredictable. Maciejewska-Potapczykowa et al. (5) were the first to report organogenesis from callus produced by stem pieces of cucumber, but they did not describe the methods for obtaining shoots. Alsop et al. (1) obtained only callus from several organ explants with various concentrations of NAA (1-naphthaleneacetic acid) and BA (6-benzylamino purine). However, some bud-like knobs were observed in callus grown at 0.1 mg/1 NAA and 0.1 mg/1 BA. Aziz et al. (2) also described bud-like nodules on callus derived from internode pieces of cucumber, but they could induce only root formation.

Others working with cucumber have been able to produce adventitious buds and/or shoots from either hypocotyls or cotyledons (5, 9, 10, 11). Cotyledons appear to be the better explant for use in organogenesis experiments (4, 7, 11). However, callus from cotyledons is characterized by proliferation of fibrous roots whereas callus derived from hypocotyls is not (9, 10). Other organs have also been used as explants.

The main objective of this research was to increase the efficiency of shoot production from cotyledonary explants by manipulating growth regulator concentrations in the medium and determining the optimum cotyledon age for regeneration.

Explant source. For the time course study, seeds of two cultivars of cucumber ('Straight 8' and 'Sumter') were surface sterilized on a gyratory shaker at 100 rpm. Seeds were soaked in a 50% Clorox (2.6% NaOCl) solution for 30 minutes followed by 5 rinses in sterile distilled water. Ten seeds each were placed in 100 × 15 mm plastic petri plates containing 1% Bactoagar that had been autoclaved at 121C for 15 minutes. Plates were sealed with Parafilm and placed in darkness at 30C for seed germination.

For the secondary media study, seeds of the breeding line Gy 14A were surface sterilized in the same manner as described previously. After placing the sterilized seeds on water agar as described above they were germinated in the dark at 30C for 5 days.

Regeneration procedure. For the time course study, cotyledons were excised from each seedling at 2, 4, 6, 8 or 10 days of age. Five 2 × 2 mm explants from the same cotyledon were placed adaxial side down in 100 × 15 mm plastic petri plates containing 20 ml of a Murashige-Skoog (MS) (8) medium with 1% agar and supplemented with 3% sucrose, 1 mg/1 NAA and 1 mg/1 BA. The medium (designated ORG) was adjusted to pH 5.8 prior to autoclaving. Five plates were used for each temperature, day and cultigen (cultivar or breeding line) combination.

Cultures were maintained at 22C under a 24 hour photoperiod of fluorescent and incandescent lamps. Cultures were transferred to the same medium after 4 weeks. Data on callus diameter and numbers of roots and shoots were recorded after 4 and 8 weeks. The experiment was a split-plot treatment arrangement in a randomized complete block design with two replications. Data were taken as means of 5 petri plates.

For the secondary media study, cotyledons from 5-day-old seedlings were excised and divided into six 2 × 2 mm pieces. Explants were placed adaxial side down into 100 × 15 mm plastic petri plates containing 20 ml of ORG and placed under the same environmental conditions outlined above. After 4 weeks, all of the new growth was removed from the explants and transferred to ORG where the cultures remained for 4 additional weeks. The 8-week-old tissue was then placed onto MS medium containing 16 combinations of NAA and BA (concentrations were 0, 0.1, 0.3, and 1 ppm each of NAA and BA in a factorial design) where it remained for two 4-week subcultures.

Ratings on regeneration, and number of roots and shoots per plate were recorded after 4 weeks and again after 8 weeks on the 16 NAA-BA treatment combinations. The regeneration rating, based on color and differentiation of the tissue was as follows: 0=brown, 3=undifferentiated green tissue, 5=green nodular tissue, 7=green nodular tissue with leaves, 9=green tissue with shoots. The experiment was a split-plot treatment arrangement (with cultigen as whole plot and NAA-BA combination as subplot) in randomized complete block design with 2 replications. Data were taken as means of 5 petri plates.

Time course study. Seeds began germinating after 2 days on water agar. Cotyledons emerged from the seed coats after 4 days for 'Sumter' and 5 days for 'Straight 8'. A hard, green, nodular tissue began forming around the cut edges of the cotyledon pieces after about 1 week, and adventitious shoots began forming from this tissue after 3 to 4 weeks on the culture medium. The number of days from germination to explanting (seedling age) affected callus growth, and root and shoot regeneration. Shoot production decreased for the 8 and 10 day treatments and was highest (60%) from 6-day-old tissue.

It appears that 4 to 6 days is the optimum germination period for shoot regeneration from cotyledon tissue at a germination temperature of 30C. After 2 days, the cotyledons had not yet emerged from the seed coat, making it difficult to excise the explants. After 8 to 10 days in culture, the cotyledons lost their regenerative ability.

Secondary Media Study. Shoot formation occurred infrequently for all secondary media treatments and did not occur until 6 to 8 weeks after the explants had been on the secondary medium. One reason for the poor regeneration may have been a slight browning of the tissue which occurred after 8 weeks. The cultures probably needed to be transferred more frequently than every 4 weeks.

Total number of shoots per plate was influenced mainly by the concentration of NAA in the secondary medium (Table 2). When NAA was absent, Gy 14A developed shoots at all BA concentrations except 3 mg/1. A medium with 0.3 mg/1 BA and no NAA produced the greatest number of shoots. The best regeneration ratings were also on secondary media lacking NAA (Table 2).

Root production was also influenced by NAA and BA levels. Numerous, short, callus-covered roots developed on all media having NAA but lacking BA (Table 2). BA tended to depress total root production, especially at the higher NAA concentrations. BA also appeared to promote root elongation.

One piece of tissue on a medium with 0.0 mg/1 NAA and 0.3 mg/1 BA produced a number of abnormal bipolar structures that began to differentiate shoots and roots. All of the embryoids were abnormal and none grew into plantlets. These results were promising since previous studies had never yielded more than 2 shoots from a single explant. The structures also appeared to have arisen indirectly from a friable yellow tissue which is typical of embryogenesis. It is also possible that some of the shoots observed on other plates may have actually been embryos that remained attached to maternal tissues. This would partially explain why shoots developed after 12 to 16 weeks instead of the 4 to 8 weeks described in the previous experiment. These possibilities led us to change our focus to regeneration through embryogenesis which had been reported previously in cucumber (6)

Literature Cited

1. Alsop, W. R., W. W. Cure, G. F. Evans and R. L. Mott. 1978. Preliminary report on in vitro propagation of cucumber. Cucurbit Genet. Coop. Rpt. 1:1-2.
2. Aziz, H. A., B. H. McCown and R. L. Lower. 1986. Callus initiation from cucumber (Cucumis sativus L.) fruits. Cucurbit Genet. Coop. Rpt. 9:3.
3. Bouabdallah, L. and M. Branchard. 1986. Regeneration of plants from callus cultures of Cucumis melo L. Z. Pflanzenzucht. 96:82-85.
4. Custers, J. B. M. and L. C. Buijs. 1979. The effects of illumination, explant position, and explant polarity on adventitious bud formation in vitro of seedling explants of Cucumis sativus L. cv. Hokus. Cucurbit Genet. Coop. Rpt. 2:2-4.
5. Maciejewska-Potapczykowa, W., A. Rennert and E. Milewska. 1972. Callus induction and growth of tissue cultures derived from cucumber plant organs of four different sex types. Acta. Soc. Bot. Poland 41:329-339.
6. Malepszy, S. and A. Nadolsky-Orczyk. 1983. In vitro culture of Cucumis sativus. I. Regeneration of plantlets from callus formed by leaf explants. Z. Pflanzenphys. 111:273-276.
7. Moreno, V., M. Garcia-Sogo, I. Granell, B. Garcia-Sogo and L. A. Roig. 1985. Plant regeneration from calli of melon (Cucumis melo L.) cv. Amarillo Oro. Plt. Cell Tiss. Organ cult. 5:139-146.
8. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
9. Novak, F. J. and M. Dolezelova. 1982. Hormone control of growth and differentiation in the in vitro cultured tissue of cucumber (Cucumis sativus L.). Biologia 37:283-290.
10. Sekioka, T. T. and J. S. Tanaka. 1981. Differentiation in callus cultures of cucumber (Cucumis sativus L.). HortScience 16:451 (Abstr.).
11. Wehner, T. C. and R. D. Locy. 1981. In vitro adventitious shoot and root formation of cultivars and lines of Cucumis sativus L. HortScience 16:759-760.

Table 1. Root and shoot production of cotyledons excised at 2 day intervals over a 10-day period from 2 cultigens of cucumberz.
		Cotyledon
		growth	No. per plate:		% explants with:
Day	Cultigen	(mm)	Shoots	Roots	Shoots	Roots
2	Straight 8	17.7	1.3	3.0	26.7	60.0
	Sumter	15.3	1.0	2.0	20.0	43.3
4	Straight 8	15.8	0.4	1.6	8.0	32.0
	Sumter	17.0	0.8	2.0	15.0	40.0
6	Straight 8	14.3	3.0	0.3	60.0	6.7
	Sumter	13.5	1.0	0.5	20.0	10.0
8	Straight 8	15.5	0.5	0.5	10.0	10.0
	Sumter	15.8	0.0	0.2	0.0	4.0
10	Straight 8	13.3	0.0	0.0	0.0	0.0
	Sumter	12.0	0.0	0.0	0.0	0.0
LSD (5%)		2.1	1.0	1.4	21.5	28.5
		15.2	0.7	1.1	15.1	22.7
CV (%)		14	111	102	112	99
zData are means of 5 plates taken after 8 weeks on MS media with 1 mg/1 each of NAA and BA.

Table 2. Shoot and root numbers from Gy 14A cucumber 8 weeks after being subcultured from MS with 1 mg/1 each of NAA and BA to 16 media with different combinations of NAA and BAz.
NAA	BA	Friability	Regeneration	No. per plate:
Concn.	Concn.	Ratingy	Ratingx	Shoots	Roots
0.0	0.0	3.2	3.3	0.3	3.5
	0.3	4.3	6.7	4.0	4.5
	1.0	5.3	3.8	0.5	2.2
	3.0	5.0	4.0	0.0	1.5
0.3	0.0	7.3	3.0	0.0	2.8
	0.3	6.0	3.2	0.0	2.2
	1.0	6.0	3.6	0.9	0.8
	3.0	5.7	4.3	0.0	3.7
1.0	0.0	8.9	4.2	0.4	18.8
	0.3	6.9	4.3	0.0	4.4
	1.0	7.3	4.0	0.0	2.2
	3.0	5.6	2.6	0.0	4.2
3.0	0.0	9.0	3.0	0.0	24.1
	0.3	8.0	3.5	0.0	0.5
	1.0	7.7	3.4	0.0	1.3
	3.0	6.8	3.4	0.0	3.4
LSD (5%)		1.1	2.5	1.1	1.6
		5.8	3.6	0.2	5.7
CV (%)		14	35	394	75
zConcentrations were 0.0, 0.3, 1.0, and 3.0 mg/1 each of NAA and BA in a factorial design.
YFriability was rated 1 to 9 (1hard, 5moderately friable, 9very friable).
XRegeneration was rated 1 to 9 (1brown tissue, 3undifferentiated green tissue, 5tissue with buds, 7tissue with leafy structures, 9tissue with shoots).