This document discusses the in vitro propagation of bamboo through tissue culture techniques. It describes the selection of nodal explants from juvenile bamboo shoots, sterilization procedures, and various initiation, multiplication, rooting, and hardening media compositions that produced the best results. The highest rates of shoot bud induction and multiplication were achieved using MS media supplemented with BAP and KIN plant growth regulators. Root induction was maximized with IBA or NAA in the media. Hardening involved transferring plantlets to soil mixtures in the greenhouse, resulting in 92.5% survival rates. Tissue culture is presented as an efficient method for large-scale bamboo propagation.

1. ASSIGNMENT
TOPIC: IN VITRO PROPAGATION OF BAMBOO
Submitted to
Dr. Sneha Macwana Submitted by
Associate Professor 67 - Rahul Chandera
Department Of Plant Breeding & Genetics 68 - Kirpal Chaudhary
B. A. College Of Agriculture 70 - Amit Kanani
Anand Agricultural University, Anand 80 - Ajay Solanki

2. Content of Presentation
1) Introduction
2) Selection of explant
3) Sterilization
4) Initiation Medias
5) Multiplication Medias
6) Rooting Medias
7) Hardening
8) References

3. 1) Introduction :
 Bamboo is the strongest and fastest-growing perennial grass species and is
unique with complex branching patterns; woody culms and gregarious,
monocarpic flowering plant.
 Botanical name : Bambusa vulgaris, (common bamboo)
 Family : poaceae
 Sum family : Bambusoideae
 Chromosome no. 2n = 24

4. Cont..
 More than 1,439 species of bamboo categorized under 115 genera are found
in the world, For bamboo different propagation techniques are available, such
as seed propagation, clump division, rhizome and culm cuttings .
 But these methods suffer from serious drawbacks for large or mass scale
propagation, have long flowering cycle (30–120 year) which is a limiting factor
for large scale planting programme.

5. Cont…
 When required for planting programmes, For mass scale propagation (> 500 000
plants per year) classical techniques are largely insufficient and inefficient, and
tissue culture is the only viable method.
 Bamboo being the fastest growing plant, generates over 60 tons of oxygen every
year from one acre of bamboo garden which is enough for breathing over 200
human being. Apart from reduction of CO2 by its carbon sequestration ability.
 It is one of the best commercial plantations either as a pure crop or mixed crop
for the farmers to generate additional income from land that are not suitable for
regular agriculture.
 India has larger bamboo forests, making up nearly 13 % of the country’s forest
area.

6. Cont..
 Beema Bamboo is a new variety which has got huge potential to bring
revolution in the field of Bio-Energy.
 Beema bamboo has 65% digestible cellulose which can be processed into
Ethanol. Under the available cellulosic ethanol process, 4 Kg of bamboo can
be made to produce 1 Kg of Ethanol. Thus, cultivation of bamboo under
Precision Farming can generate 10,000 to 12,000 lit of Ethanol per acre per
year
 Bamboo is popularly referred to as ‘‘poor man’s timber’’, ‘green gold’,
‘‘wonder plant’’.
 The annual production of bamboo in India is about 4.6 million tonnes, about
1.9 million tonnes is used.

7. What is Plant Tissue Culture
 Plant Tissue Culture is a technique of growing plant cells, tissues,
organs, seed or other plant parts in a sterile environment on a
nutrient medium.

8. Plant Tissue Culture depends upon
 Totipotency : Totipotency is the genetic potential of a plant cell
to produce the entire plant. In other words, totipotency is the cell
characteristic in which the potential for forming all the cell types in
the adult organism is retained.
 Plasticity : Plasticity is the abilities of plants to change their
metabolism, growth, development… in order to best suit their
environment.

10. 2) Selection of explants:
 Young and juvenile shoots were collected from nursery raised 2-3 years
old plants.
 The nodal segments with single axillary buds were used as explant for
micro propagation.

11. 3) Sterilization
 Start laminar & disinfect surface with alcohol.
 Wash Bamboo Cutting with normal tap water.
 Cutting regular Stem of Bamboo with one bud in each cutting stalk.
 Treatment of fungicide with Bavistine.
 Treatment of Agcl2 For 8 min.
 Wash with disttiled water 2 -5 times
 Cut Both the end of explant.
 Put explant in to test tube.
 Seal the test tube & Write details.

13. 4) Initiation Media
1.Media composition : MS+1mg/L BAP+0.2mg/L NAA+ 3% Sucrose+0.8%
Agar
Name Of the crop : Bamboo
Total quantity : 1000 ml
Stock Quantity
Stock A (20x) 50 ml
Stock B (100x) 10 ml
Stock F (100x) 10 ml
Stock G (100x) 10 ml
Inocitol 100 mg
KH2PO4 170 mg
Sucrose 30 gm
Initial Volume 500 ml

14. Cont…
PGR Quantity
BAP 5 ml
NAA 1 ml
Final volume 1000 ml
pH of media 5.65
Agar 8 gm
Media per test tube 12 ml
Total test tube filled 83

15. 2.Medias for Nodal explants of Bambusa balcooa:
 1) MS with 4.4 mg/lit. BAP, 0.53 mg/lit. NAA and 1% sucrose
 2) MS with 16.11 mg/lit. NAA and 1% sucrose
 Results: Shooting multiplication, rooting & 92.5% hardening.
3. Medias for nodal explants of B. vulgaris:
"BAP 5.0 mg/lit.+ NAA 4.0 mg/lit
 "Result: Mass multiplication

16. Observation after 1 Week

17. 5) Multiplication medias:
 For Shoot bud multiplication, the regenerated shoot buds were cultured
on MS medium augmented with various concentrations of BAP and/or
KIN (0.5–5.0 mg/lit.) alone and/or in combination with 0.5 mg/lit. KIN
and BAP.
 Cultures were transferred to fresh media at 3 weeks interval for further
shoot bud proliferation.
 The regenerated shoots were separated from the multiple shoot clumps
and were transferred to elongation medium.

18. Cont…
 The excised shoot buds were cultured on MS medium
supplemented with different concentrations of gibberelic acid GA3
(0.5–3.0 mg/lit.) in combination with 3.0 mg/lit BAP and/or 4.0
mg/lit KIN for shoot bud elongation.

19. Cont..
 The highest frequency (91.5 %) of multiple shoot bud induction with
maximum number of shoots (85 shoots/explant) was noticed on MS
medium with 3.0 mg/l BAP and 0.5 mg/l KIN.
 The regenerated multiple shoots were elongated on MS medium with 4.0
mg/l KIN and 2.0 mg/l gibberellic acid (GA3) give maximum shoot length
(4.9 cm).
 Best shoot multiplication was found in media containing BA 1.5 mg/l. The
newly developed shoots were recoreded in 5-8 numbers whose shoot
length were 3.8±0.4 observed.
 Best period for recycling of multiplication shoots was recorded i.e. 18 days
old culture. Result was observed by Mudai and Borthaker 2009.

20. 6) Rooting:
 After completion of shoot multiplication and elongation cycle, the
elongated shoots ([2–3 cm in length)were transferred to MS medium
supplemented with different concentrations of indole-3 butyric acid
(IBA)(0.5–5.0 mg/lit) alone and/or in combination with 0.5 mg/lit BAP.
 For rooting shoots produced roots within 3 weeks of culture were
recorded.
 The highest percent of root induction (75 %) was observed on MS medium
fortified with 2.0 mg/lit IBA and 0.5 mg/lit KIN combination and the
maximum number of roots obtained was 5.0 roots/shoot .

21. Cont…
 The percent of rooting was increased with increasing the concentrations
of IBA up to 2.0 mg/lit, while the rooting was declined with further
increase the IBA concentration in the medium.

22. Cont…
 For root induction developed shoot bunch (3-4 shoots) were placed
in the media containing different concentration of NAA (0.5-1.5
mg/l).
 Best result was recorded (4±1 roots) in media containing 1.5 mg/l
NAA whose length was 2-3 cm recorded after 2-3 weeks. Results
were observed by Arya 2002 and Pratibha and Sharma, 2011.

23. 7)Hardening & Acclimatization
 Hardening is lab to land transfer technology . It provides healthy
growth of the plant under milder conditions before the plant
exposed to environmental
Two type of Hardening
1. Primary Hardening
2. Secondary Hardening

24.  Primary hardening : The In vitro Plantlets raised after pre-hardening
were removed from culture jars, washed thoroughly under disttiled
water remove traces of medium from the roots.
 Plantlets were transferred to hardening trays. One set of hardening tray
was consist of 3:1 ratio of coco peat and vermicompost.
 Secondary hardening : Afterwards the plants are transferred to polybags
filled with potting mixture and grown under shaded house for 6 – 8
weeks. This is called Secondary hardening. After secondary hardening
the plants are suitable for growing in farmer’s fields

25. Cont..
 For hardening, the in vitro rooted plantlets were first washed with distilled
water to remove medium to prevent contamination.
 The washed plantlets planted into plastic pots filled with mixture of
Coco peat with vermicompost ( 3:1 ratio by volume) and transferred in to
greenhouse at temperature 24±2 C˚ with relative humidity 60-70%.
 The survival rate was 92.5% and 87.5% after 30 and 60 days of hardening,
respectively .

26. Cont…
 During hardening stage some plantlets were found wilting in the first
three days of transferring and some leaves were dried up and
subsequently detached from the shoots.
 This may be due to unrestricted loss of water from their leaves or low
hydraulic conductivity of roots and root-stem connections .
 Therefore, just after transferring the pots were covered with clear
polyethylene with 2-3 small holes for first five days.

27. Cont…
 After ten day of hardening two-three new leaves were developed from each
shoots. Gradually the plantlets started growing and the leaf number
increased as the plant height increased.
 The plants were shifted to open shade house condition. In shade house the
plants were further transferred into bigger polybags and were irrigated with
water.
 In next two months these plants developed rhizome in polybags condition in
the shade house. Than after plants are ready to field plantation.

28.  References
 Hoot Through Nodal Explants Culture Of Bambusa Bambos For Biomass Production; P.
Muthukumaran, N. Saraswathy, S. Abarna, R. Kanthimathi, V. Monisha, N. Niranjana Devi,
 The Use Of Tissue Culture Technique For Propagation Of Bamboo (Dendrocalamus Giganteus
Wallich); Mona A-amin And Essam El- Atrach Timber Trees Dep., Hort. Res. Inst., A.R.C. Egyp.
 Callus And Cell Suspension Culture Of Bamboo Plant; Phyllostachys Nigra Shinjiro Ogita
 Tissue Culture Strategies For Genetic Imprin Vitro Propagation Of The Giant Bamboo
Dendrocalamus Giganteus; Munromd. Aktar Hossain,bayezid Mahmud Khan,mohammad Azhar
Uddin
 Development Of An Efficient Protocol For Plant Regeneration From Nodal Explants Of Recalcitrant
Bamboo (Bambusa Arundinacea Retz. Willd) And Assessment Of Genetic Fidelity By DNA Markers;
K. Kalaiarasi ,P. Sangeetha ,S. Subramaniam ,P. Venkatachalam
 Biotechnology Of Bamboos; Pooja Thapa, Devinder Kaur, Priyanka Sood, Rupali Mehta, Jasmine
Brar
 Protocol For Induction Of Multiple Sovement Of bamboo; J. Gielis, H. Peeters, K. Gillis, J.
Oprins,p.C. Debergh.
 Rapid Multiplication Of Bamboo By Tissue Culture; A.L.Nadgir, C.H.Phadke,p.K.Gupta