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Cucurbit Genetics Cooperative Report 22:5-7 (article 3) 1999

Transfer of Cucumber (cucumis sativus L.) Plantlets Regenerated from in vitro Culture

Hongwen Cui

Department of Horticulture, Northwestern Agricultural University, Yangling Shaanxi, 712100, P.R. China

Yuxiang Yuan

Institute of Horticulture, Henan Academy of Agricultural Sciences, Zhengzhou, 450002, P.R. China

When plantelets grow in an environment of constant temperature, high humidity, low light intensity and sterile surroundings, they exhibit a decrease in leaf photosynthesis rate, slow growth, thin leaf epidermis, reduced ability for stomatal opening and closure, poor water-controlling functions, and incomplete physiological and morphological function of the root system. Since their adaptability to adapt to an outside environment is poor, they require a transitional stage before transplanting (plantlets) to the open field. to date, there are many successful reports of plantlets being regenerated from cucumber in vitro culture, and there are also reports regarding the transfer of plantlets to pots (2,3,4). However, there is little detailed information about survival rte of plantlets after transfer to pots. Cai Rongqi (1) developed a transfer method named "T2 medium rerooting" where plantlets derived using fertilized ovules are transferred during in vitro culture. The plantlet survival rate using this technique was 93.3%. The aim o four work was to test the effect of different transfer substrates, temperature treatment after planning, and plantlet size on survival rate after plantlet transfer to pots this would allow for a determination of optimal conditions during transfer.

Methods. rooted plantlets were regenerated during many subcultures of shoot tips or axillary buds from the cucumber breeding line 'Chang-176'. These were used as experimental materials. Films sealed on the top of vessels were removed and unsealed vessels were placed in a growth chamber maintained at 25+ 1C. with a 16-hour photoperiod provided by cool-white fluorescent light at 1500 lux. Uniform plantlets were "hardened-off" for 3 days before transfer to plastic pots. subsequently, rooted plantlets were taken from vessels, agar was washed off of their roots with tap water, and plantlets wee then transferred to plastic pots (diameter - 10 cm) containing different substrates. Plastic pots were put onto the enamel plate, and water was added to ensure constant humidity.

Three experiments (Exp. 1-3) were designed. In Exp. 1., four different substrates [vermiculite (A); fine sand:vermiculite = 2:1(B), fine sand:vermiculite:culture soil containing a mixture of 7:3 manure to soil] were compared to evaluate the effect of a transfer substrate on survival rate of plantlets after transfer.

Expt. 2 was designed to identify an optimal temperature after plantlets were transferred. Vermiculite was used as the transfer substrate. There were four constant temperature treatments, varying at 5 C intervals among 15-30 C, and one variable temperature treatment where plantlets were subjected to 15 C for 3 days and then 20 C for 7 days. Duration for each treatment was 10 days. Plantlets were acclimatized for 3 days under room temperature prior to transplanting.

In Exp. 3, plantlets were classified as small, medium and large. Plantlets of different sizes were transferred to plastic pots containing vermiculite. The experiment was conducted at 15 C for 3 days and then at 20 C for 7 days.

All experiments above were conducted in a growth chamber with strict temperature control. The transfer survival rte was recorded after 13 days when new leaves were produced by the plantlets. The "U-test" was used for percentage's determination.

Results. For Experiment 1, the number of rooted plantlets that survived in the plastic pots containing different substrates are presented in Table 1. Among the four transfer substrates, vermiculite was optimal (96.7% survival rte) and differed significantly from the other three substrates tested. Cultured soil was unfavorable for transfer survival of rooted plantlets. Vermiculite had the best air permeability followed by fine sand containing vermiculite. Culture soil is rich in microbial and manure, but rooted plantlets did not grow well in this media. It was observed that rot at the junction between the root and the stem was the main reason for the low survival.

For Experiment 2, data of the effect of various temperature treatments on survival rate after transfer indicated that differential survival rates occurred among plantlets (Table 2.). Comparative analysis of data from temperature intervals between the 15-30 C, nd constant temperature treatments, indicated survival rate decreased with increasing temperature and that significant differences existed between the various temperature treatments. When given the changeable temperature treatment of 15 C for 3 days and then at 20 C for 7 days, plant survival was as high as 100%. This treatment was superior to all other values. Compared with treatments at 25 C and 30 C under constant temperature, the variable temperature treatment increased plant survival by a factor of 2.4 times and 10 times, respectively. These results can be explained by the fact that with increasing temperature transpiration increased, and water loss and nutrition waste likely increased. Plantlets for such treatments were unadaptable to the open field environment. Lower and changeable temperature treatments gradually improved the adaptability of plantlets to survive in an open field environment.

Since the results of the second experiment indicated that lower and changeable temperature could significantly improve survival rate after transfer, we used this temperature treatment in the Experiment 3. The results of the transfer of plantlets of different sizes are presented in Table 3. Results indicated that large plantlets having more than 5 leaves and 6.1 cm in height) survive remarkably well when transferred to the plastic pots (highest survival rate), while small plantlets had difficulties surviving.

Transplantation of plantlets into the open field results in different survival rates depending on treatment. Plants in plastic pots that had treatment survived treatment were put outside the room for 3 days to acclimatize to the outer environment. Plantlets were taken out of the plastic pots, and part of the root-bound substrate was removed. These plantlets were then transplanted into the open field. the survival rte after transplanting was as high as 92.9%. There was no difference in leaf morphology, growth habit, flower and fruit characteristics among those plantlets.

Conclusions. In conclusion, the data indicate that: (1) The highest survival rate could be obtained when rooted plantlet were transferred to the plastic pots containing vermiculite; (2) lower and changeable temperature (at 15 C for 3 days and then at 20 for 7 days) provided the highest survival rate after transfer to plastic pots (as high as 100%); and (3) the survival rate of large plantlets with more than 5 leaves and 6.1 cm in height was higher than smaller plantlets. Plantlets that survived the initial transfer were successfully transplanted into the open field (survival rates up to 92.9%). These plants flowered and set fruit.

In order to ensure a well-developed root system it is important to select a suitable transfer substrate. Our results showed that vermiculite is best transfer substrate among four substrates used. The use of lower and variable temperatures increased survival rate. this rate was slightly higher than the T2 medium rerooting method put forward by Cai Rongqi (1998). The former procedure, however, does not include root pruning, which in our case saved 507 days rerooting time and simplified the transfer process. Therefore, we believe our method is simpler, more convenient, and more practical than that proposed by Cai Rongqi.

Table 1. Effect of different transfer substrates on survival rate of the transfer of cucumber plants to pots.

Transfer substrate
No. of plantlets transferred
No. of plantlets surviving
Rate of survival (%)
A
30
29
96.7 a
B
30
14
46.7 b
C
32
14
43.8 b
D
28
4
14.3 c

z The different letter indicated that the results of treatment are significantly different by U-C test at p = 0.01.

Table 2. Effect of temperature treatments on survival rate of the transfer of cucumber plants to the pots.

Temperature treatment (˚C)
No. of plantlets transferred
No. of plantlets surviving
Rate of survived (%)
Notex
15
30
25
83.3 b
Constant
15 - 20
28
28
100.0 a
Changeable
20
28
18
64.3 c
Constant
35
33
14
42.4 d
Constant
30
28
3
10.7 e
Constant

Table 3. Effect of plantlet size on survival rate of cucumber plants transfer to pots.

Size of plantlet y
No. of plantlets transferred
No. of plantlets surviving
Rate of survival (%)
Small
16
6
40
Medium
16
12
75
Large
14
14
100

Plantlets were classified as 1) small (less than 3 leaves and lower than 3.5 cm in height); 2) medium (3 to 4 leaves and 3.5 to 5.0 cm in height), and 3) large (more than 5 leaves nd higher than 5.1 cm in height).

Literature Cited

  1. Cai Rongqi. 1992. Study on fertilized ovule in vitro culture in cucumber (Cucumis sativus L.) Tianjin Agricultural sciences 1:14-16.
  2. Chee, P.P. 1990. High frequency of somatic embryogenesis and recovery of fertile cucumber plants. HortScience 25L792-793.
  3. Misra, A.K. and S.P. Bhatnagar. 1995. Direct shoot regeneration from the leaf explant of cucumber (cucumis sativus L.). Phytomorphology 45:47-55.
  4. Punja, Z.K., Abbs, N. Sarmento, G. G. and F.A. Tang. 1990. regeneration of Cucumis sativus var. sativus and C. sativus var. hardwickii, c.melo, and C. metuliferus from explant through somatic embryogenesis and organogenesis: Influence of explant source, growth regulator regime and genotype. Pl. Cell Tissue and Organ Cult. 21:93-102.
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Page citation: Wehner, T.C., Cucurbit Genetics Cooperative;
Created by T.C. Wehner and T. Ng, 1 June 2005; design by C.T. Glenn;
send questions to T.C. Wehner; last revised on 15 December, 2009