International Journal of Chemical Studies 2019; SP6: 427-430
P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2019; SP6: 427-430
(Special Issue -6)
3 rd National Conference
Dipti Shradha Tirkey On
St. Xavier’s College, Mahuadanr,
Latehar, Jharkhand, India
PROMOTING & REINVIGORATING AGRI-HORTI,
TECHNOLOGICAL INNOVATIONS
Diwakar Prasad Nirala
Faculty of Forestry, Birsa [PRAGATI-2019]
Agricultural University, Ranchi, (14-15 December, 2019)
Jharkhand, India
Phallo Kumari
PG, Deptt. of Botany, Vinoba Micropropagation from nodal explants of rose
Bhave University, Hazaribagh,
Jharkhand, India (Rosa hybrida L.) at different concentration of
BAP (6-Benzyl Amino Purine)
Dipti Shradha Tirkey, Diwakar Prasad Nirala and Phallo Kumari
Abstract
The nodal explants from young healthy shoots were cultured on basal medium of BAP (6-Benzyl Amino
Purine) containing several concentrations. It was reported as most effective growth regulator in
stimulating shoot proliferation of many plant species. The aim of the study was to analyze the
effectiveness of different concentration of BAP (6-Benzyl Amino Purine) in micropropagation of rose
(Rosa hybrida L.). The maximum percentage of number of explants forming shoots (95%) was observed
in 3 mg/l concentration of BAP (6-Benzyl Amino Purine). The shoot elongation was considerably slow.
Findings could be utilized as an efficient tissue culture technique to yield large number of shoots from
nodal explants of roses.
Keywords: Rose, Explants, BAP, Shoots etc.
Introduction
Rose is one of the most important commercial crops of genus Rosa that belongs to family
Rosaceae. Roses have been one of the world’s most popular ornamental plants for a long time.
They are grown worldwide as cut flowers and potted plants and in home gardens. The flowers
vary greatly in size, shape and colour. It is generally propagated by vegetative methods like
cutting, layering, budding and grafting. Although propagation by vegetative means is a
predominant technique in roses, yet it does not ensure healthy and disease-free plants.
Moreover, dependence on season and slow multiplication rates are some of the other major
limiting factors in conventional propagation. Significant features of in vitro propagation
procedure are its enormous multiplicative capacity in a relatively short span of time;
production of healthy and disease free plants; and its ability to generate propagules around the
year (Bhojwani, 1981) [1]. Tissue culture derived dwarf roses used for pot plant production
have a faster rate of growth, early flowering, and exhibit shorter shoots and more laterals than
conventionally produced plants.
Materials and Methods
Plant materials
Nodal explants containing lateral buds of actively field-grown Rosa hybrid L. var. ‘Perfume
Delight’ rose were used for multiplication experiments. Each node contains an area of
Corresponding Author: meristematic tissue called an axillary bud. They were cut in 3-4 cm length segments.
Dipti Shradha Tirkey
St. Xavier’s College, Mahuadanr, Explants Sterilization
Latehar, Jharkhand, India For the sterilization, the explants were first washed thoroughly in running tap water for 10-15
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minutes. After that they were again washed with liquid contaminants are blown away by the ultraclean air and
detergent. After washing with detergent, explants were again thereby an aseptic environment is maintained over the
washed with running water to remove traces of detergent for working area.
10-15 minutes. These washing explants were dipped in 70% b) pH meter- It is necessary for the measurement and
ethyl alcohol for 30 seconds. After alcohol dip, explants were adjustment of pH of the nutrient medium.
surface sterilized with freshly prepared 0.1% Mercuric c) Autoclave- It is very important for sterilization of
chloride (HgCl2) for 5 minutes. After that, explants were nutrient media, glass goods, instruments etc.
thoroughly washed for 3-4 times with sterile water to remove d) Hot air oven- It is necessary for drying the washed glass
any traces of mercuric chloride. goods.
Culture Media Lab goods: Test tubes, Measuring cylinder, Conical flasks,
Murashige and Skoog’s (1962) [7] medium (MS) was used for Pipettes, Beaker, Petri dishes were well washed and then
rose propagation in semi solid form. dried in hot air oven. Scalpel, Forceps, Scissors, Cotton plugs
(wrapped in gauge cloth) were also used during the
Composition of MS micropropagation.
All the equipments were first sterilized in autoclave under
Macronutrients Micronutrients steam at a pressure of 15lb/in² and a temperature of 120˚C for
MgSO₄7H₂O 370 mg/l AlCl₂ 0.025 mg/l 15 minutes. The semisolid media (Murashige and Skoog,
KH₂PO₄ 170 mg/l H₃BO₃ 6.2 mg/l 1962) [7] was used with BAP and coconut milk. Explants
KNO₃ 1900 mg/l MnSO₄4H₂O 22.3 mg/l (Rosa hybrida L.) were cultured to the sterilized medium and
NH₄NO₃ 1650 mg/l ZnSO₄7H₂O 8.6 mg/l culture tubes were capped with sterilized cotton plugs.
CaCl₂ 440 mg/l Na₂MoO₄2H₂O 0.25 mg/l Inoculation is done under laminar air flow very carefully. All
Vitamins CuSO₄5H₂O 0.0025 mg/l the equipments are used is sterilised. The explants are
Thiamine HCl 0.1 mg/l KI 0.83 mg/l inoculated in semisolid media. After inoculation, culture tubes
Pyridoxine 0.5 mg/l FeSO₄7H₂O 27.85 mg/l are kept in the culture room and observed every day. Cultures
Myoinositol 100 mg/l Na₂EDTA.2H₂O 37.25 mg/l
were maintained at 25 ± 3˚C air temperatures in a culture
Glycine 2 mg/l Chemicals required
room. After 2 weeks the explants were examined for axillary
Thiamine HCl 0.1 mg/l BAP (6-Benzyl Amino Purine)
shoot growth.
Carbon source Coconut milk
Sucrose 30 m/l
Results and Discussions
Final Preparation of semi solid media: All the chemicals Observation
were added in distilled water. Media are made semi solid by The percentage shoot formation was varied among the
using solidifying agent agar-agar powder (8%) was added in different concentration of BAP (6-Benzyl Amino Purine) as
the liquid media and pH (5.8) was checked. It was boiled till shown in Table-1. The development of axillary shoot from the
the homogenous was obtained. Finally, the media was nodal explants was observed after 9 to 14 days. There
sterilized by Millipore filter. infections were also observed during the culture. The
presence of cytokinin in the culture medium helped in the
Lab instruments multiplication of shoots in hybrid roses and auxin help in
a) Laminar air flow – It is the most suitable, convenient multiplication of roots.
and reliable instrument for aseptic work. All
Table 1: Observation of growth of explants (BAP conc. 3mg/l)
Days of observation No. of cultures No. of culture showing infection Observation (growth of explants) Callus Shoot growth
3 days 20 0 No growth 0 0
1 week 20 4 No growth 0 0
10 days 16 4 Callusing 0 0
2 week 12 0 Axillary shoot growth 9 9
20 days 12 0 Shoot growth 11 11
3 weeks 12 0 Shoot developed 12 12
Table 2: Effects of different concentration of BAP in the shoot proliferation
Concentration of BAP (mg/l) No. of cultures No. of explants No. of explants forming shoots (%)
1 20 20 40%
2 20 20 65%
3 20 20 95%
6 20 20 45%
9 20 20 0%
The formation of shoots was observed from nodal explants of Table-2. No any shoot formation was observed in the
Rose (Rosa hybrida L.) at different concentration of BAP (6- concentration of 9 mg/l. Thus the concentration 3 mg/l of
Benzyl Amino Purine). Shoots formation were taken place BAP (6-Benzyl Amino Purine) with MS media was best for
from nodal side of explants after 20 days of culture. The multiplication and it was selecting for culturing of nodal
maximum percentage of number of explants forming shoots explants for production of large number of shoots. Tissue
(95%) was observed in the concentration of 3 mg/l followed culture system in roses has been established (Hsia and
by 2 mg/l (65%), 6 mg/l (45%), and 1 mg/l (40%) as shown in
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Korban, 1996; Ibrahim and Debergh, 2001; Kim et al., 2003; Hameed et al., 2006; Drefahl et al., 2007) [4, 5, 6, 3, 2].
After 3 weeks of initial culture, nodal explants containing developed shoots. BAP was the most effective growth
lateral buds cultured on MS medium in the experiment regulator in stimulating shoot proliferation.
Initial stage callusing Callusing
Growth- emergence of shoot Emergence of multiple shoots (20 days)
Growth after 30 days Growth after 36 days
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Conclusion
From the above results, it can be concluded that the maximum
percentage of number of explants forming shoots (95%) was
observed in 3 mg/l concentration of BAP (6-Benzyl Amino
Purine). It was the most effective concentration for growth
regulator in stimulating shoot proliferation.
References
1. Bhojwani SS. A tissue culture method for propagation
and low temperature storage of Trifolium repens
genotypes. Physiol. Plants. 1981; 52:187-190.
2. Drefahl A, Quoirin MG, Cuquel FL. Micropropagation of
Rosa hybrida cv. Vegas via axillary buds. Acta
Horticulturae. 2007; 751:407-411.
3. Hameed N, Shabbir A, Ali A, Bajwa R. In vitro
micropropagation of disease free rose (Rosa indica L.).
Mycopath. 2006; 35:38.
4. Hsia CN, Korban SS. Organogenesis and somatic
embryogenesis in callus cultures of Rosa hybrid and Rosa
chinensis minima. Plant Cell Tissue and Organ culture.
1996; 44:1-6.
5. Ibrahim R, Debergh PC. Factors controlling high
efficiency of adventitious bud formation and plant
regeneration from in vitro leaf explants of roses (Rosa
hybrida). Scientia Horticulturae. 2001; 88:44-57.
6. Kim SW, Oh SC, In DS, Liu JR. Plant regeneration of
rose (Rosa hybrida) from embryogenic cell-derived
protoplasts. Plant Cell Tissue and Organ Culture. 2003;
73:15-19.
7. Murashige T, Skoog F. A revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Physiol. Plant. 1962; 115:493-497.
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