THE INFLUENCE OF FACTORS TO THE REGENERATION OF RED RASPBERRY
(RUBUS ALCEAEFOLIUS POIR.)
Trinh Thi Huong1, Do Dang Giap2, Tran Trong Tuan2*
1
Tay Nguyen Institute for Scientific Research - Vietnam Academy of Science and Technology
2
Institute of Tropical Biology - Vietnam Academy of Science and Technology
*
Email: [email protected];
1. SUMMARY
Red raspberry is a highly economical plant, but difficult to propagate. As a result, red
raspberry cultivars have to be imported and that leads to a significant increase in the product’s
price. This study aimed to perfect the micro-propagation process of red raspberry and enhance
the cultivar resources for local farmers. The results showed that tender stem nodes were
sterilized with HgCl2 0.1% for 12 minutes gave the highest survival rate and regeneration of
explants (75%). After 6 weeks culture, the most suitable medium to multiply shoots was MS
(Murashige and Skoog, 1962) supplemented with 0.5 mg l-1 BA, 20 g l-1 sucrose, 8 g l-1 agar,
pH=5.8 and 41.7 mg l-1 FeSO4 (increase three fold FeSO4 content in MS medium). The best root
formation rate of shoot was obtained on ½MS medium supplemented 1.0 mg l-1 IBA, 20 g l-1
sucrose, and 8 g l-1 agar. The high rate of survival seedlings was obtained (85%) when
transferred to ex vitro.
Key words: BA, FeSO4, HgCl2, IBA, micropropagation, red raspberry
2. INTRODUCTION
Red raspberry (Rubus alceaefolius Poir.) is of the Rose family (Rosaceae), grows as vines,
hashairas well as thorn on its branches,simple leaf and the hairy leaf blade is palmate (5 lobes).
Flowers grow in inflorescences and have five white petals. Red raspberry is very popular in
North America and several Asian countries. The whole plant is medicinal; for example, the
leaves are useful in improving blood circulation, reducing internal body heat and helping with
stasis and inflammation. Moreover, red raspberry leaves contain calcium, niacin, manganese,
potassium and vitamins such as vitamin A, B, C and E. Red raspberry fruits not only are
delicious but also comprises a lot of vitamin C and antioxidants. In addition, comprised in the
fruits is a great concentration of potassium and vitamin K which can improve the digestive
system, help healing wounds, enhance protectionagainst cancer, maintain eyesight, strengthen
bones, increase cell quantity, moderate urinary system diseases, treat kidney stones and diabetes,
prevent morning sickness and heighten sexual prowess.
In temperate countries, red raspberry is a plant of high commercial value and used tobreed
hybrids with interesting colors such as yellow and purple. However, in Vietnam, a tropical
country, red raspberry is not as popular sincebeing a temperate fruit. Not cultivated, red
raspberry rather grows wildly and especially with popularity in the Northern forest hills. Those
wild red raspberries are low not only at productivity but also in nutrition contents, in comparison
to the farmed ones in temperate countries. Now, one kilogram of red raspberry can be sold for
VND 200,000 – 250,000. Because of the discussed nutritious as well as economic values, several
regions in Lam Dong Provincehave started to cultivate red raspberry. However, the price of
imported cultivars stays relatively high, at around VND 60,000 – 70,000 per seedling. After 3 or
1
5 years, the seedlings will be degenerated and need to be replaced. In addition, the traditional
methods of propagation are inefficient: the cuttings’ roots tend to get rotten in the process.As a
consequence, the application of in vitro technique in micropropagation not only is essential to the
cultivar resources for local red raspberry farmers but also helps reducing the cost of importing
this plant’s seedlings. Hence, this study aimed to perfect the micropropagation process of red
raspberry and enhance the cultivar resources for local farmers, which subsequently would benefit
the final product price of red raspberry.
3. MATERIALS AND METHODS
3.1. MATERIAL
Explants source of experiment is young stem nodes/internodes of one-year-old France red
raspberries (Rubus alceaefolius) which cultivated at Da Lat farm.
3.2. METHODS
SHOOT REGENERATION STAGE
The effect of sterilization solution and sterilization time on regeneration ability of ex vitro
explants
Internodes of 5 - 6 cm in length with 1 to 2 dormant shoots, leaves and petiole that were
dissected and sterilized with 70% alcohol for 30 second then rinsed three to five times with
sterile distilled water. Internodes were sterilized with various concentrations of HgCl2 and
Ca(OCl)2, which added with one to two drops of Tween 20 for various period of time in
following step. At last, explants rinsed three to five times with sterile distilled water again.
Sterilized explants were cultured on MS basal medium (Murashige and Skoog, 1962) [6],
with 0.6 mg l-1 BA, 0.1 mg l-1 IBA, 30 g l-1 sucrose, 8 g l-1 agar and pH 5.8 (Zawadzka and
Orlikowska, 2006) [7].
The effect of TDZ on shoot regeneration from in vitro leaves
The disinfected leaves that were detached from shoots were cultured on MS medium
(Murashige and Skoog, 1962) [6] containing various concentrations of TDZ (0.5, 1.0, 1.5, 2.0 mg
l-1).
The effect of BA on shoot regeneration for internodes
The disinfected internodes were cultured on MS medium (Murashige and Skoog, 1962) [6]
containing various concentrations of BA (0.5, 1.0, 1.5, 2.0 mg l-1).
SHOOT MULTIPLICATION STAGE
The effect of BA on shoot multiplication of in vitro shoot
Nodal segments, about 1 - 2 cm in length from 6-week-old in vitro shoots were transferred
to MS medium with 20 g l-1 sucrose, 8 g l-1 agar, pH 5.8 and different concentrations of BA.
REGENERATION PLANTS AND ACCLIMATIZATION
The effect of FeSO4 on in vitro shoot quantity
One to two cm long internodes from in vitro shoots were cultured on MS medium
supplemented with 20 g l-1 sucrose, 8 g l-1 agar, 0.5 mg l-1 BA and various concentrations of
FeSO4 to treat yellows problem.
2
The effect of IBA on root formation
Shoots in vitro, about 2 - 3 cm in length were transferred on root induction medium
containing 20 g l-1 sucrose, 8 g l-1 agar and IBA with different concentrations
The effect of mineral content on root formation
Shoots in vitro, about 2 - 3 cm in length were transferred on various mineral media to
determine suitable medium for root formation. All media containing 20 g l-1 sucrose, 8 g l-1 agar
and IBA.
Acclimatization
6-week-old plantlets with well developing root system were transferred to chamber within
2 weeks then were established in soil in nursery.
STATISTICAL ANALYSIS
Data were recorded and test by Microsoft Excel 2007 and Duncan’s multiple range test
(Duncan, 1955) [1] at 5% level of Statgraphics Centurion XV software package.
4. RESULTS AND DISCUSSION
4.1. SHOOT REGENERATION STAGE
The effect of sterilization solution and sterilization time on regeneration ability of ex vitro
explants
In sterilization experiment using Ca(OCl)2, all explants were bacterial contamination and got
0% in survival rate (table 1). Because explants have hair as well as thorn on its branches, so it is
hard to sterilize. The effect of Ca(OCl)2 10% could not decontaminate all of contaminated
agents. At increased sterilization time (15 mins), contamination rate decreased to 91.67% but
stem nodes became brown instead of shoot regenerated. Ca(OCl)2 10% was not suitable for
sterilization of red raspberry. Thus, the authors studied the effect of HgCl2 0.1% on shoot
regeneration of red raspberry. HgCl2 was actually harmful for human and microbiology but plant
tissues. Necrosis rate of plant tissues when treated by HgCl2 was lower than treated by other
sterilization solutions such as Ca(OCl)2, javel… This experiment showed that the effect of HgCl2
was better than the effect of Ca(OCl)2 (table 2).
Table 1. The effect of Ca(OCl)2 10% on shoot regeneration
Sterilization
time (mins)
3
6
9
12
15
18
Contamination rate
Internode
Leaf
Petiole
*
100b
100c
100d
100b
100c
100d
100b
100c
96.67c
100b
96.67b
94.33b
91.67a
88.33a
86.33a
92.33a
89.67a
85.33a
Survival rate
Internode
Leaf
0
0
0
0
0
0
0
0
0
0
0
0
Petiole
0
0
0
0
0
0
*Means in the same column that are followed by different letters are significantly different (p ≤ 0.05) using Duncan’s Multiple Range Test
Table 2. The effect of HgCl2 0.1% on shoot regeneration
Sterilization
time (mins)
Contamination rate
Internode
Leaf
Petiole
Survival rate
Internode
Leaf
Petiole
3
3
6
9
12
15
18
100.00a*
76.67b
48.33c
23.33d
15de
6.67e
100.00a
78.33b
46.67c
18.33d
16.67d
8.33e
100.00a
76.67b
45.00c
18.33d
13.33de
8.33e
0.00e
18.33d
41.67b
78.33a
43.33b
31.67c
0.00e
16.67d
41.67b
76.67a
43.33b
33.33c
0
0
0
0
0
0
*Means in the same column that are followed by different letters are significantly different (p ≤ 0.05) using Duncan’s Multiple Range Test
In this study, contamination rate of petiole was the lowest and contamination rate of
internode was the highest. Because stem nodes have hair as well as thorn on its branches so it is
hard to sterilize. Petiole has no thorn and number of hair on petiole was not as many as number
of hair on leaf or internodes, hence contamination rate of petiole was lower.
When sterilization time got more than 12 minutes, sterilization rate was decreased but
explants were necrotized. According to this experiment, explants were sterilized with HgCl2
0.1% solution for 12 minutes got the highest survival rate (75%).
Petioles died all after 2-week-cultured. Shoot formation from dormant buds of internodes
and callus initiation from leaves were happened. After 4 weeks, axillary buds had a strong
development (figure 1a), callus induction were high desirable (figure 1b).
The effect of TDZ on shoot regeneration of in vitro leaves
After 4 weeks of cultured, the result showed that shoots were induced on the MS medium
supplemented with TDZ. Shoots formation from callus were initiated within 3 weeks. Shoot
formation was low and shoot pattern was vitrification (figure 1c). Shoot could develop normally
when transferred into new medium. At increased TDZ level (1.0 - 1.5 mg l-1), vitrification in
shoot increased although shoots were transferred to new medium.
Table 3.The effect of TDZ on shoot formation of in vitro leaves
TDZ (mg l-1)
0.0
0.5
1.0
1.5
2.0
Number of
shoots/explant
0.00d*
1.67c
3.67b
4.33ab
4.67a
Explants morphology
No shoot stimulation
Small shoot, vitrification
Small shoot, vitrification
Stronger shoot, vitrification
Stronger shoot, vitrification
*Means in the same column that are followed by different letters are significantly different (p ≤ 0.05) using Duncan’s Multiple Range Test
The effect of BA on shoot regeneration for internodes
Table 4. The effect of BA on shoot formation of in vitro internodes
Number of
BA s (mg l-1)
Explants morphology
shoots/explant
0.0
0.00d*
No shoot stimulation
0.5
1.67c
Small shoot, vitrification
1.0
3.67b
Small shoot, vitrification
1.5
4.33ab
Stronger shoot, vitrification
2.0
4.67a
Stronger shoot, vitrification
*Means in the same column that are followed by different letters are significantly different (p ≤ 0.05) using Duncan’s Multiple Range Test
4
Conclusion of shoot regeneration stage
In shoot regeneration stage, shoot formation via internodes was more efficient than shoot
formation via in vitro leaves. Moreover, shoot formation via internodes was stronger and
vitrification was not happened. Hence, internode was suitable explants for shoot regeneration
stage.
4.2. SHOOT MULTIPLICATION STAGE
The effect of BA on in vitro shoot multiplication
The effect of BA on shoot proliferation of raspberry was reported by McNicol et al. (1990),
Hoepfner et al. (1996), Mezzetti et al. (1997) and Graham et al. (1997) [4, 3, 5, 2]. The result
(table 5) shown that BA stimulated proliferation of red raspberry.
Table 5. The effect of BA on in vitro shoot multiplication
BA concentration
(mg l-1)
0.00
0.25
0.50
0.75
1.00
Number of shoot/explant
Shoot morphology
0.00d*
5.33c
8.67a
7.00b
6.33b
No shoot stimulation
Strongly shoot, light-yellow leaves
Strongly shoot, light-yellow leaves
Strongly shoot, light-yellow leaves
Strongly shoot, light-yellow leaves
*Means in the same column that are followed by different letters are significantly different (p ≤ 0.05) using Duncan’s Multiple Range Test
This result was resemble result of McNicol et al. (1990), Hoepfner et al. (1996), Mezzetti et
al. (1997), and Graham et al. (1997) [4, 3, 5, 2]. At increased BA level, number of shoot per
explant increased. Especially, shoot cultured on medium containing 0.5 mg l-1 BA developed
average 8.67 shoots (table 5, figure 1d). In the opposite direction, on PGR-free medium, shoot
formation did not happen. On medium supplemented 0.5 mg l-1 BA, shoot was not as long as
other BA concentrations. Because of shoot formation on medium containing 0.5 mg l -1 gave the
best number of shoot, thus nutritional completion happened strongly.
In this experiment, although shoot developed strongly, light-yellow leaf syndrome was
happened (figure 1d, e). The hypothesis was reported iron deficiency effected the development
of shoots.
4.3. REGENERATION PLANTS AND ACCLIMATIZATION
The effect of FeSO4 on shoot multiplication of in vitro shoot
Iron played an important role in plant regeneration. Iron was catalytic agent in chlorophyll
formation and iron also carried oxygen. Iron deficiency caused some symptoms include leaves
turning light-yellow or brown. Nevertheless, with the increased in FeSO4 level (2 times more or
27.8 mg l-1 instead of 13.9 mg l -1), leaves became greenish. Light-yellow leaf syndrome was
repaired if FeSO4 concentration increased 3 to 5 times more (41.7 to 69.5 mg l-1) and shoots
possessed more large green leaves (table 6, figure 1f). Zawadzka and Orlikowska (2006) [7]
reported the attendance of iron via FeEDDHA in medium plays an important role in plant
regeneration of fivespecies of raspberry. However, with the increased FeSO4 level (4 to 5 times
more or 55.6 to 69.5 mg l-1), number of shoot decreased. Therefore, the increased FeSO4 level
with three times is the most suitable for regeneration of shoots.
5
Table 6. The effect of FeSO4 on shoot multiplication of in vitro shoot
FeSO4 concentration
Number of
Shoot morphology
(mg l-1)
shoot/explant
Strongly, light-yellow
13.9
7.33d
leaves
Strongly, light-yellow
27.8(2 times more than in MS medium)
7.67cd
leaves
41.7 (3 times more than in MS medium)
10.33a
Strongly, green leaves
55.6 (4 times more than in MS medium)
8.67b
Strongly, green leaves
69.5 (5 times more than in MS medium)
8.33bc
Strongly, green leaves
*Means in the same column that are followed by different letters are significantly different (p ≤ 0.05) using Duncan’s Multiple Range Test
The effect of IBA on root formation
On PGR-free medium, root initiation did not happen. On medium supplemented with 0.5 mg
l IBA, 1 - 2 roots were formed and got 1.0 - 1.5 cm in length but secondary root induction did
not happen yet. Shoot on medium containing more than 1 mg l-1 got higher rooting rate (90%)
and developed 3 - 4 roots included secondary roots. However, plantlets with 1.0 - 1.5 cm in
length root (figure 1m) developed better when transferred in soil. Thus, shoot on medium
supplemented with 1 mg l-1 IBA get the best root morphology.
-1
Table 7. The effect of IBA on root formation
IBA concentration
(mg l-1)
0.0
Rooting
rate (%)
0.00c*
0.5
73.33b
1.0
1.5
2.0
90.00a
93.33a
96.67a
Root morphology
No formation
Strongly, 1 - 2 root, 0.5 - 1 cm in length, no secondary root
formation
Strongly, 3 - 4 roots, 1 - 1.5 cm in length, secondary roots
Strongly, 3 - 4 roots, 2.5 - 3 cm in length, secondary roots
Strongly, 3 - 4 roots, 4 - 5 cm in length, secondary roots
*Means in the same column that are followed by different letters are significantly different (p ≤ 0.05) using Duncan’s Multiple Range Test
The effect of mineral content on root formation
Mineral was important with development and growth of explants. In rooting stage, macromineral was reduced to help plantlets adjusted to poor nutritional condition before transferred to
nursery.
Shoot on ½MS (MS medium with half of macro-mineral) medium got the best root
formation rate (100%) (Figure 1i). When macro-mineral was doubled (2MS), rooting rate
decreased to 50% though shoots were larger and possessed more new leaves (Figure 1h).
Medium
½MS
MS½
MS
2MS
Table 8.The effect of mineral content on root formation
Root formation
Root morphology
(%)
100.00a*
Strongly seedling, 4 - 5 roots, 1 – 1.5 cm, lateral roots
91.67b
Strongly seedling, 4 - 5 roots, 1 – 1.5 cm, lateral roots
85.00c
Strongly seedling, 4 - 5 roots, 1 – 1.5 cm, lateral roots
46.67d
Strongly seedling, 4 - 5 roots, 1 – 1.5 cm, lateral roots
*Means in the same column that are followed by different letters are significantly different (p ≤ 0.05) using Duncan’s Multiple Range Test
6
Six-week-old plantlets were taken out of medium (Figure 1g) and were soaked in weak
fungicide solution for 5 minutes. Plantlets eventually were established in soil in nursery and a
high survival rate (85%) was obtained within 4 weeks (Figure 1n).
Figure 1. Micropropagation of red raspberry. a. Shoot formation from ex vitro internode; b. Ex vitro leaf cultured
after 4 weeks; c. Shoot formation from in vtro leaf on MS medium with 1.0 mg l-1 TDZ; d, e. Shoots on MS medium
with 0.5 mg l-1 BA, narrows show light-yellow shoots; f. Shoots on MS medium with 0.5 mg l-1 BA and 41.7 mg l-1
FeSO4; g. Shoots on rooting medium containing 1.0 mg l-1 IBA; h. Plantlets on 2MS medium with 1.0 mg l-1 IBA; i.
Plantlets on ½MS with1.0 mg/l IBA; k. Shoot on MS medium with 2.0 mg l-1 IBA; m. In vitro plantlets before
transferred to chamber; n. 4-week-old plantlets in chamber.
5. CONCLUSION
Tender stem nodes were sterilized with HgCl2 0.1% for 12 minutes gave the highest survival
rate and regeneration of explants (75%).
The most suitable medium to multiply shoots was MS (Murashige and Skoog, 1962)
medium supplemented with 0.5 mg l-1 BA and 41.7 mg l-1 FeSO4 (increase three fold FeSO4
content in MS medium).
1
The best root formation rate of shoot was achieved on ½MS medium supplemented 1.0 mg lIBA.
The high rate of survival seedlings was obtained (85%) when transferred to greenhouse.
7
Acknowledgement: Authors are grateful to the Institute of Tropical Biology, VAST for the
financial support to carry out the present experiment.
REFERENCES
[1]. Duncan D. B., 1955. Multiple range and multiple F-tests. Biometrics, 11: 1-42.
[2]. Graham J., Iasi L., Millam S., 1997. Genotype-specific regeneration from a number of
Rubuscultivars. Plant Cell Tiss. Org. Cult., 48: 167-173.
[3]. Hoepfner A. S., Nestby R. and Nybom H., 1996. Genetic deviation initiated by
adventitious shoot regeneration from tissue cultured red raspberry. J. Hort. Sci., 71:71–79.
[4]. McNicol R. J. and Graham J., 1990. In vitro regeneration of Rubusfrom leaf and stem
segments. Plant Cell Tiss. Org. Cult., 21: 45-50.
[5]. Mezzetti B., Savini G., Carnevali F. and Mott D., 1997. Plant genotype and grrowth
regulators interaction affecting in vitro morphogenesis of blackberry and raspberry. Biol.
Plant., 39 (1): 139-150.
[6]. Murashige T. and Skoog F., 1962. A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol. Plant., 15: 473-497.
[7]. Zawadzka M. and Orlikowska T., 2006. Factors modifying regeneration in vitro of
adventitious shoots in five red raspberry cultivars. J. Fruit Ornam. Plant Res., 14: 105-115.
ẢNH HƯỞNG MỘT SỐ YẾU TỐ LÊN SỰ TÁI SINH CÂY RED RASPBERRY (Rubus
alceaefolius Poir.)
Trịnh Thị Hương1, Đỗ Đăng Giáp2, Trần Trọng Tuấn2*
1
Viện Nghiên cứu khoa học Tây Nguyên, VAST
2
Viện Sinh học Nhiệt đới, VAST
*
Email: [email protected];
Tóm tắt
Red raspberry là loài cây trồng có giá trị kinh tế cao nhưng rất khó nhân giống. Do đó,
giống cây red raspberry phải nhập khẩu nên cây giống có giá thành cao. Nghiên cứu này nhằm
mục đích hoàn chỉnh qui trình vi nhân giống red raspberry và tăng cường nguồn cung cấp giống
cho nông dân địa phương. Kết quả cho thấy, các đốt thân được khử trùng bằng HgCl 2 0,1% trong
12 phút cho tỉ lệ sống và tái sinh cao nhất (75%). Sau 6 tuần nuôi cấy, môi trường thích hợp để
nhân chồi là môi trường MS (Murashige và Skoog, 1962) có bổ sung 0,5 mg/l BA, 20 g/l
sucrose, 8 g/l agar, pH = 5,8 và 41,7 mg/l FeSO4 (cao gấp 3 lần hàm lượng FeSO4 trong môi
trường MS). Tỉ lệ chồi mẫu hình thành rễ tốt nhất trên môi trường ½ MS bổ sung 1,0 mg/l IBA,
20 g/l sucrose và 8 g/l agar. Tỉ lệ cây con sống sót cao nhất khi chuyển ra vườn là 85%.
Từ khóa: BA, FeSO4, HgCl2, IBA, red raspberry, vi nhân giống,
8