Nguyen Thi Tam et al Tjtp chi KHOA HQC & CONG NGHg 82(06):103-108
EVALUATION O F C O L D T O L E R A N C E OF XUAN C H A U H U O N G , Q5, C27, KHANG DAN, U17 A N D N H I U U 63 RICE C U L T I V A R S USING IN VITRO C U L T U R E T E C H N I Q U E
Nguyen Thi Tam, Tang Thi Ngoc Mai, Chu Hoang Mau*
College of Education - TNU SUMMARY
In recent years, the world in general and Vietnam in particular have experienced abnormal climate changes.
The Northem mountainous area, the Red River Delta and Northem Central regions are often affected by cold weather that prolonged with temperature getting lower and lower. This is the major cause of the decreasing rice yield in these regions, in which winter-spring rice is most seriously influenced by low temperature, especially at seeding stage. Then, studying cold tolerance ability and enhancing resistance to low temperature to improve productivity and stabilize yield of rice breeds are practically required for these regions. In this paper, we announce the results of evaluating cold tolerance at callus level of six rice cultivars: Khang Dan, Xuan Chau Huong, Nhi Uu 63, C27, U17 and Q5 using in vitro culture technique to create the initial materials for selection of cold-tolerant rice lines. Results showed that after cold treatment at different time thresholds (1, 3, 5, 7, 9, 11, 13 and 15 days), callus tissue of all six rice cultivars have different rate of survival, growth rate and regeneration capacity, of which the highest are of Xuan Chau Huong. Finally, 86 callus and 210 rice lines have been created and serving for further research.
Key words; Callus, cold tolerance, in vitro, Oryza saliva, plant regeneration
INTRODUCTION
In Vietnam, rice {Oryza sativa) production plays an important role in the national economy. The Northern mountainous area, the Red River Delta and Northem Central regions are the main rice raising areas, which, however, are usually impacted by cold weather. In winter in recent years, temperature in these regions often goes down very low (under lO^C) for a long fime, having a serious influence on rice yield and quality, especially winter-spring rice crops.
Therefore, as concerned by many authors such as [1], [2], [3], and [5], researches on cold- tolerant ability and improvement of chilling- resistant ability of rice cultivars in order to increase and stabilize rice production in cold condition are very necessary. To assist scientists to find out good cold-tolerant rice cultivars, we conducted an assessment on cold tolerance at callus level of six rice cultivars including Khang Dan, Xuan Chau Huong, Nhi Uu 63, C27, UI 7 and Q5 using in vitro culture technique.
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MATERIALS AND METHODOLOGY Materials
Research materials are grains of six rice cuhivars consisting of Khang Dan (KD), UI 7 provided by Thai Nguyen Department of Agriculture and Rural Development; Xuan Chau Huong (XCH), Nhi Uu 63 (NU63), C27 supplied by Bac Kan Department of Agriculture and Rural Development; and Q5 taken fi-om Bac Giang Department of Agriculture and Rural Development.
In vitro cultivation method
- Disinfection of rice grains: ripe rice grains were peeled hulls away and sterilized with alcohol 70%
for 1 minute, shook in Javen solution 60% for 20 to 25 minutes; then rinsed with distilled water 3-5 times.
- Callus tissue creation: the grains after disinfection were placed on basic MS medium (Murashige and Skoog, 1962), supplemented with 3% sucrose, 0.8% agar, 2.4 D 2 mg/l, pH = 5.8.
Each culture bottle consisted of 10 to 15 grains.
The cultures were placed in dark for a week, then
Nguyen Thi Tam et al Tap chi KHOA HQC & CQNG NGH$ 82(06): 103 108 under ambient light in the culture room with
2000-lux intensity for two weeks. After that, we assessed the ability of callus induction.
Callus treatment: formed callus tissues were divided into 3 mm, placed in modified MS medium consisfing of MS, sucrose 3%, agar 0.8%, 2.4D 1 mg/l, pH = 5.8 and kept at 5"C ± 0.5°C in fridges based on different fime phases (1,5,9, 11, 13, 15 days).
Plant regeneration: Callus ttissues after cold treatment were transferred to fresh MSR medium including MS, BAP 2mg/l, sucrose 3%, aga 0.8%, pH = 5.8 to regenerate plants. Survival rate and growth pace were assessed after 3 weeks, then after 8 weeks, their regeneration ability would be evaluated
- Complete plant development: green shoots were separated into different lines and grown to maturity in MS medium with MS, sucrose 3%, aga 0.8%, NAA 0.2mg/l, pH = 5.8 with a density of 10 green shoots every bottle under Neon light at 2,000-lux intensity.
FINDINGS AND DISCUSSION
Exploration of callus generation ability and growth rate
In order to assess adaptation of rice cultivars in tissue culture system in the aims of cold-tolerant assessment and cell type selection in the future, we conducted experiments to observe ability of callus induction, growth rate and plant regeneration capability of studied cultivars.
After 3 weeks in culture room, callus tissues were evaluated, then cut into parts of three mm^
and grown in regeneration medium. Relative
growth rate of the callus ttissue was calculated by the number of callus units (1 callus unit = callus portion size 3 mm^). Regeneration ability was assessed after 8 weeks and showed results in table 1.
All experimental cultivars were able to induce callus and regenerate plants. However, rate of callus formation and growth of different types were not the same. In fact, callus tissues of C27 had the most ability of induction and development (98.59%) while those of Nhi uu 63 had the least capability. On the other hand, plant regeneration of C27 was the least (8.54%);
whereas, that of Xuan Chau Huong was the most (15.39%).
Many researches indicate that, plant regeneration depends on genome of each cultivar [4], [6], [7], [8]. By experiments, we found that dry hard yellow-white tissues had more regeneration ability than wet dark-brown ones. Then, all studied rice cultivars are capable of callus induction and plant regeneration, which are suitable for following researches relating to in vitro culture method.
Impact of cold on survival rate of callus tissues To build a process of selecting cold-tolerant rice cultivars, it is necessary to define cold-tolerant thresholds of callus tissues. We identified ability of bearing cold of cultivars at callus level based on criteria of cold-tolerance and plant regeneration capability of callus after treatment at 5° C ± 0.5° C in different time thresholds (1,5,9,
11, 13, and 15 days).
Table 1. Exploration of callus induction, growth rate and regeneration of rice cultivars Cultivars Rate of callus induction (%) Growth rate
(callus unit/grain/3 weeks)
Rate of plant regeneration (%) after 8 weeks U17
NLr63 KD Q5 XCH
027
96.96 ± 0.39 82.29 ± 0.33 93.41 ±0.45 93.56 ±0.34 86.96 ±0.27 98.59 ±0.53
2.72 ±0.17 1.45±0.16 1.82±0.16 2.33 ±0.11 2.31 ±0.09 2.82 ±0.10
15.13±0.12 9.17 ±0.02 13.24 ±0.03
9.46 ± 0.03 15.39±0.21
8.54 ±0.27
• , \
Nguyen Thi Tam et al Tap chi KHOA HQC & CONG NGHE 82(06):103-108 Table 2. Rate of callus survival after 3 weeks of regeneration raising
„ . . , Rateof survival (%)
1 day 5 days 9 days 11 days 13 days 15 days
U17 NU63
KD Q5 XCH
C27
94.96 ± 0.06 86.14 ±0.25 92.81 ±0.14 83.08 ±0.17 95.13 ±0.12 79.67 ±0.18
43.35 ±0.3 34.17±0.15 39.73 ±0.21 26.93 ± 0.07 45.35 ± 0.34 24.84 ±0.19
22.57 ±0.4 20.03 ±0.13
15.72 ±0.22 10.24 ±0.21 21.11±0.14 10.26 ±0.15
12.35 ±0.45 10.27 ±0.19 9.37 ±0.08 6.43 ±0.15 12.34 ±0.11
5.11 ±0.08
8.27 ± 0.23 7.15 ±0.09 6.42 ± 0.23 2.01 ±0.09 8.14±0.I2
0
8.13±0.17 6.93 ±0.12 4.86 ±0.15
0 7 .36 ± 0.35
0
Cold tolerance of callus was assessed by their survival rate after 3 weeks of regeneration growing. Results showed that after 3 weeks, living ivory-white tissues was larger than they had been before processing; by contrast, dead tissues in black, yellow or white did not change their size.
Survival rate of callus of studied cultivars after treatment at low temperature were presented in table 2.
It is seen obviously in table 2 that the more days in cold treatment, the less survival rate callus tissues were at. Cold tolerance of different cultivars was dissimilar. Rate of survival callus of U17 was highest at most of time thresholds (94.96%, 43.35%, 22.57%, 12.35%, 8.27% and 8.13% in turn at time thresholds 1,5,9,11,13,15 days). On the contrary, that of C27 was lowest (79.67%, 24.84%, 10.26% and 5.11% at time thresholds of
1, 5, 9, 11 days respectively). After 10 days, survival rate of callus of all cultivars decreased quickly. After 13 to 15 days at low temperature, callus of C27 died out; callus of, Q5 were at the same situation at the threshold of 15 days. Our research results are completely similar to previous
Table 3. Relative growth rate of callus after
Studies, which proved that processing time was opposite to survival rate [4], [8].
Impact of cold on growth rate of callus tissues In addition to assessment on survival ability of callus, we observed their development capability after cold treatment at different time phase and three weeks of restoration. All results are showed in table 3.
Table 3 points out the differences among relative growth rate of six cultivars. In details, Xuan Chau Huong had the highest rate of growth (2.92 unit/tissue, 3.12 unit/tissue, 3.21 unit/tissue, 2.20 unit/tissue 2.08 unit/tissue, 1.88 unit/tissue and 1.52 unit/tissue respectively corresponding to each time threshold), followed by U17, and the least rate is of C27. It can be inferred that the more survival rate the callus tissues had, the more growth rate they had, and vice versa. Table 3 also reveals that at the processing phase of 1 and 5, cold had positive influence on callus's growth, promoted their development, compared to controls, increased the most in U17 (3.27 unit/tissue at phase of 5 days). However, when we rose treatment time, growth rate of callus decreased gradually until they died out [8].
cold'treatment and 3 weeks of regeneration
Cultivars U17 NLTeS
KD Q5 XCH
C27
Control 2.66 ± 0.02 2.08 ± 0.02 2.48 ± 0.02 2.46 ± 0.02 2.94 ±0.04 2.88 ±0.11
Relative growth 1 day
3.15 ±0.04 3.08 ±0.11 2.80 ±0.16 2.94 ±1.14 3.12 ±0.08 2.17±0.10
5 days 3.27 ± 0.03 3.20 ±0.04 3.18 ±0.05 3.18 ±0.07 3.21 ±0.04 2.46 ±0.02
rate (callus unit/ tissue/ 3 weeks) 9 days
2.12 ±0.04 171 ±0.08 2.01 ±0.02 1.54 ±0.06 2.20 ±0.05 1.36 ±0.03
11 days 2.03 ± 0.04
1.56 ±0.05 1.81 ±0.04 1.47 ±0.03 2.08 ± 0.03 1.22 ±0.02
13 days 1.70±0.01 1.27 ±0.02 1.61 ±0.01 1.09 ±0.03 1.88 ±0.03
.
15 days 1.42 ±0.02 1.02 ±0.03 1.32 ±0.02 1.52 ±0.02
Nguyen Thi 1
Cultivars U17 NU63
KD Q5 XCH
C27
'am et al Table 4.
Iday I8.15±0.22 1I.28±0.I0 17.93 ±0.14 8.81 ±0.04 23.24 ± 0.06
8.32 ±0.10
Tap chi KHOA HQC & CONG NGH$
Shoot regeneration ability of callus isses after cold treatment
Shoot regeneration after 8 weeks of culture (%) 5 days
14.54 ±0.13 9.57 ±0.23 14.18±0.!0 12.35 ±0.14 I4.39±0,14 6.42 ±0.14
9 days I8.I5±0.22 1I.28±0.10 17.93 ±0.14 8.81 ±0.04 23.24 ± 0.06
8.32 ±0.10
11 days 16.46 ±0.29 9.34 ±0.19 16.32±0.17 6.24 ±0.14 17.12 ±0.07
1.35 ±0.07
13 days 13.86 ±0.09
9.29 ± 0.03 10.96 ±0.08 6.56 ±0.26 14.32 ±0.14
0
IS days 15.21 ±0.14 10.01 ±0.12 12.74 ±0.15
0 16.00 ±0.12
a
Shoot regeneration ability of callus tissuesafter cold treatment
Capability of regenerating shoots after 8 weeks of culture of callus tissues surviving from cold processing is shown in table 4. Results uncovers that callus tissues of most species after cold treatment at different time thresholds were still able to regenerate green shoots, especially at the phase of 1,5, and 9 days. It is inferred from table 4 that Xuan Chau Huong had the highest regeneration rate at all thresholds (23.24%, 14.39%, 23.24%, 17.12%, 14.32% and 16%
corresponding to phases of 1, 5, 9, 11, 13 and 15 days). The culfivars having the lowest ability of shoot rising was C27 (8.32%, 6.42%, 8.32%, 1.35% at 1, 5, 9, 11 days respecfively). At the phases of 13 and 15 days, callus of C27 completely died; and none of Q5 callus could be alive at the threshold of 15 days. To conclude, the more treatment time increased, the more shoot regeneration decreased. On the other hand, if tissues could survive from cold treatment, they could more easily grow shoots than their controls.
According to Le Tran Binh and his colleagues (1998), after extremely dealing with dead sensitive cells, we could select cells with higher vitality and regeneration [5]. Then, regeneration ability did not only rely on genome but also on cold treatment time. By experiments, we chose 86 tissue types and 210 lines of 6 rice culfivars having good cold-tolerance which are being grown to supply for following researches.
By the research on cold tolerance and regeneration ability of rice cultivars, we defined that cold-tolerant threshold of U17, Nhi uu 63, Khang Dan and Xuan Chau Huong was 15 days,
of C27 was 11 days, and of Q5 was 13 days.
However, for more accurate results, it is important to observe their development on fields, analyze genetics and biochemistry as well as to test cold-tolerant ability of descendant generations of the collected lines.
CONCLUSION
- Six rice cultivars all had ability of callus induction and shoot regeneration from callus.
Callus formation capability of C27 was the highest and of Nhi uu 63 was the lowest. On the other hand, C27 had the lowest possibility of regeneration while the highest ability was of Xuan Chau Huong cultivar.
- Processing callus tissues at 5°C ±°C in 1 to 5 days did not negatively affected their growth; by contrast, promote their development. However, increasing treatment time to 9,11,13 and 15 days prevented their growth.
Ability of shoot rising of callus surviving from cold treatment was better than that of their confrols at the phase of 9 days.
Cold-tolerant threshold of U17, Nhi uu 63, Khang Dan and Xuan Chau Huong was 15 days, of C27 was 11 days, and of Q5 was 13 days at the temperature of 5°C ± °C
Cold-tolerant ability of the rice cultivars was arranged in a decreasing order as follows:
XCH >U17 > KD > NU > Q5 > C27 We chose 86 fissue lines and 210 plant lines to supply for following researches.
Nguyen Thi Tam et al Tap chi KHOA HQC & CONG NGHE 82(06):103-108
B ^ ^ ^ ^ ^ ^ H H (
Figure 1. Some photos in assessment on cold-tolerance of rice
(A) Callus tissues from grains; (B) survival callus reborn after 4 weeks of regeneration; (C) green shoots regenerating from survival callus after 8 weeks
REFERENCES
[1]. Bertin P., Bouharmont J., & Kinet J. M. (1997),
"Somaclonal variation and improvement of chilling tolerance in rice: Chance in chilling-induce chlorophyll fluorescence". Crop Sci, 37, pp. 1727- 1735.
[2]. Bertin P., Kinet J. M., & Bouharmont J. (1995),
"Heritable chilling tolerance improvement in rice through somaclonal variation and cell line selection", AustJBot, 44, pp. 91-105.
[3]. Bouharmont J. & Dekeyser A. (1989), "In vitro selection for cold and salt tolerance in rice". Review of Advance in Plant Biotechnology, 1985 - 88. IMWIC
&IRRI,pp.308-313.
[4]. Le Tran Binh, Vo Thi Ngoc Diep & Le Thi Muoi (1995), "Research on cold and drought tolerance of different rice cultivars at callus level". Journal of Biology, n {I), p. 25-29.
[5]. Le Tran Binh & Le Thi Muoi (1998), Gene isolation and selection of rice lines having good resistance to unfavorable external environment, Vietnam National University Publisher, Hanoi.
[6]. Gamborg O. L. & Phillip G. C. (1995), "Basal media for plant cell and tissue culture" in: Plant Cell, Tissue and Organ Culture, Fundamental methods, Springer Heidelberg, pp. 301-306.
[7]. M. Al - Forkan, M. A. Rahim, T. Chowdhury, P.
Akter & L. Khaleda (2005), "Development of highly in vitro callogeenesis and regeneration system for some salt tolerance rice cultivars of Bangladesh", Biotechnology 4 (3), pp. 230 - 234
[8]. Nguyen Thi Tam (2004), Research on heat- tolerance and selection of heat-tolerant rice cultivars by plant cell technology. Biological Ph.D. thesis, Institute of Biotechnology, Hanoi.
Nguyen Thi Tam et al Tgp ch( KHOA HQC & CONG NGHg 82(06): 103 - 108
T6M TAT
DANH GlA KHA N A N G CHJU Li^NH CUA CAC G I 6 N G LUA XUAN CHAU HU'OfNG, Q5, €27, KHANG DAN, UI7 VA NHJ UtJ 63 B A N G KY THUAT NUOI C A Y IN VITRO Nguyen Thj Tam, TSng Thj NgQC Mai, Chu Hoang Mau*
Tru&ng Dgi hgc Suphgm - DH Thdi Nguyen Trong nhOng nftm gm day, khi hau the gi6i n6i chung \k Vi|t Nam n6i rieng dang co nhung bien doi bSt thudng. Vung mien nui phia BSc, dong bang song Hong v4 BSc Trung Bp la nhirng khu vuc thuong xuyen chju anh huong ciia Ignh. NhiJng nSm gan day nhift dp xu6ng rat thap \k th&i gian k^o dai ban, day chinh la nguyen nhan I^m giam nSng suat v^ san lu(jng lua gao cua vCing. Liia vu dong xuan chju tic dgng manh nhdt cua nhift dp thap, dSc bi^t a giai dogn mg, vi vay, nghien cuu kha ndng chju lanh \k tSng cuong kha ndng chdng chju v6i nhift dp thap nhSm nang cao ndng su4t v^ on djnh san luprng ciia cac giong lua la yeu cau cap thiet doi v6i cic tinh mien nui phia BSc vi khu vyc Bic Trung Bp hien nay. Trong bai bao nay, chung tdi trinh bay k^t qua ddnh gii kha ning chju lanh a mure dp mo s^o cua 6 giong lua: Xuan chau hirong, Q5, C27, Khang dan, U17 vi Nhj uu 63 bing ky thuat nuoi cay in vitro nhim chpn tao vat lieu khoi dau phuc vu chpn dong chju lanh ciia liia. Ket qua cho thay, xu ly mo seo a nhiet dp 5°C ± O.S^C voi cic nguong thai gian (1,5,9, II, 13 vi 15 ngiy), mo s?o cic giong nghien ciiu co ty le song sot, toe dp sinh truong vi khi ning tii sinh choi khic nhau, giong Xuan chau huong co ty le song sot, toe dp sinh truong vi khi nang tii sinh choi li cao nhit. Ket qua da tao dupc 86 dong mo vi 210 dong cay xanh dang trdng ngoii dong rupng phuc vu cho cic nghien cuu tiep theo.
Tir khoa: In vitro, mo seo, Oryza sativa, tdi sinh cdy, chiu Ignh.
Tel: 0913 383289