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Case study report on Sugarcane

Submitted by : Arun NID Number: 008078

Module: D2D001 School of Bioscience, Nottingham University

Recent advances in research on plant tissue culture of Sugarcane

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Sugarcane is one of the most important crops in the world.

Brazil is the largest sugarcane grower, they grow it for:

Sugar – Production of food & alcoholic beverages

Ethanol – Gasoline + ethanol blend as fuel

In sugarcane, micropropagation is important for rapid

multiplication of elite genotypes/clones and for the

spreading of new varieties.

Research attention on tissue culture of sugarcane was

intensified due to its economic importance as a cash crop.

Sugarcane tissue culture

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1. In vitro plant regeneration using shoot tip culture.

2. In vitro micropropagation through callus culture.

3. Somaclonal variation – screening for salt tolerance.

Recent research study on

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Explants used are: Young leaves Shoot tips Meristem cells Leaf sheath

Technique 1 Duration Technique 2 Duration

• Excised explants - • Running tap water 20min

• Fungicide (carbendazine @ 0.1%) 1min • Fungicide (bavistin @ 0.2%) 10min

• Bactericide (Streptomycin @ 0.1%) 1min • Distilled water wash -

• 80% ethanol 45 sec • 70% ethanol 30sec

• 3wash Double distilled water - • Mercuric chloride @ 0.1% 5min

• 3wash Distilled water -

Common surface sterilization techniques are followed:

Steps highlighted in green color are carried out inside the flow chamber

Laboratory details

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In vitro micropropagation of Sugarcane through callus cultureBehera et al Karim et al

L. CV-Nayana Isd-16 Isd-28

Explant Young meristem Leaf Sheath Leaf Sheath

Callus Induction 2.5mg/L – 2,4-D 3.0mg/L – 2,4-D 3.0mg/L – 2,4-D

Rate of callus formation 100% 90% 100%

Regeneration 2.0mg/L BAP + 0.5mg/L NAA 1.0mg/L BAP + 0.5mg/L IBA 1.0mg/L BAP+0.5mg/L IBA

No. of shoot per explant 12.4±1.90 12 15

Avg. length of shoot (cm) 6.2±0.37 4.5 5.2

Rate of shoot formation 92% 92% 100%

Rooting medium3.0mg/L NAA 3.0mg/NAA 3.0mg/NAA

Half strength MS Medium Half strength MS Medium Half strength MS Medium

No. of root per shoot 11.2±1.5 11 16

Avg. length of root (cm) 4.0±0.94 4.0 5.0

Rate of rooting 85% 85% 97%

For best callus induction - medium is supplemented with 10% coconut water.

In vitro shoots are inoculated on half-strength MS medium for better rooting.

2,4-D conc. 2.5-3.0mg/L is best for callus induction.

NAA 3.0mg/L along with ½ strength MS medium is good for rooting.

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Biradar et al Baksha et alVariety Isd – 28 Coc - 671Explants Shoot tips Shoot tips

Regeneration BAP 2.0mg/L

+IBA 0.5mg/L

BAP 2.0mg/L

Rate of shooting 75% 79.64%Rooting NAA 5mg/L NAA 2.0mg/LRate of rooting 85% 70%

In vitro plant regeneration using shoot tip culture

For shoot regeneration, cytokinin BAP was more effective than Kn and

IBA.

Auxin is essential for root initiation.

NAA is best for rooting. Whereas, IAA or IBA produce poor quality

roots.

Rate of multiplication is high in adventitious shoots.

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Plants regenerated from meristem (shoot tip) are very similar,

both

Phenotypically

Genotypically, to the parent plant.

Shoot tip culture is better than leaf roll culture for plant

production.

For shoot regeneration the combination of auxin and cytokinin

is essential.

Regeneration potential of callus was specific and genotype

dependent and also parallel with hormonal conc. and

combination.

In vitro propagation of Sugarcane

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In vitro callus regeneration and plant establishment

(1) Callus regeneration in MS+2.5mg/l 2,4-D (2) Callus regeneration in MS+2.5mg/l NAA (3&4) Multiple shoot emergence from callus tissue in MS+2.0 mg/l BAP+0.5mg/l IBA (5) Micro shoots rooted in 1/2MS+NAA(2.5 mg/l).(6) Hardening of rooted plant lets in plastic trays.

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The term somaclonal variation was first introduced by Scow Croft and Larkin (1981)

In Saccharum Sp somaclonal variation is termed as sub-clonal variation.

Salt tolerant plants were produced by tissue culture techniques Khan et al., (2004) Explant – young leaf Regeneration medium –

Fe-EDTA 1mg/L, Casein Hydrolyzate 500mg/L, Cystine free base 30mg/L

Somaclonal variation

Characteristics Soma-clones Result Source plants Result

Tiller plant 2-12 ✓ 2-7 X

Stem height (cm) 131 – 302 ✓ 136 – 261 X

No. of node per stem 10 – 20 ✓ 10 - 16 X

Brix (%) 14.70 – 17.75 ✓ 15.9 – 18.09 X

Girth of stem 6 – 8 X 7 – 9 ✓Root bandwidth (cm) 0.5 – 1.2 X 0.3 – 1 ✓

Salt tolerant soma-clones performed better in the above mentioned characteristics

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Over the past 10 years tissue culture technology of sugarcane

has been developed, but not without some challenges.

Work on Tissue culture should integrate hand in hand with

molecular techniques, it will be helpful in obtaining genetically

stable plants.

Mass propagation of Sugarcane through Tissue culture

approach will result in availability of disease free germplasm,

propagation of newer germplasm with improved agronomic

characters for the ultimate benefit of the farmers.

Sugarcane tissue culture work can be directed towards

germplasm conservation also.

Conclusion

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In order to ascertain the true to type character of the

Tissue culture raised plantlets through shoot

proliferation, it is essential to check at the genomic

level, it is felt that further experimentation is needed

towards achieving the result.

Further studies

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Alam, M. H. (1995). Highly frequency in vitro regeneration in sugarcane. Sugarcane, 6:20-21.

Baksha, R. A. (2002). In vitro shoot tip culture of sugarcane (Saccharum) variety Isd-28. Biotechnology, 1(2-4):67-72.

Begum, S. H. (1995). Efficient regeneration of plants from leaf base callus in sugarcane. Plant tissue culture, 5:1-5.

Behera, K. K. (2009). Rapid in vitro micro propagation of sugarcane (Saccharum officinarum L. Cv-Nayana) through callus culture. Nature and Science, 7(4).

Biradar, S. B. (2009). In vitro plant regeneration using shoot tip culture in commercial cultivar of sugarcane. Karnataka J. Agric. Sci., 22(1):21-24.

Heinz, D. a. (1969). Plant differentiation from callus tissue of Saccharum species. Crop Science, 9:346-348.

Karim, M. A. (2002). Micropropagation of two sugarcane (Saccharum officinarum) varieties from callus culture. Online Journal of Biological Science, 2(10): 682-685.

Khan, S. K. (2004). Somaclonal variation in sugarcane through tissue culture are subsequent screeing of salt tolerance. Asian Journal of Plant Science, 3(3): 330-334.

Liu, M. (1983). In vitro methods applied Sugarcane improvement. In: Thope T A (ed) Plant tissue cultures: methods and applications in agriculture, pp 299-323.

Maretzki, A. (1987). Tissue culture: Its prospects and problems In: Sugarcane Improvement through breeding. (Ed.) D.J.Heinz. Elsevier Science publisher B.V., 343-384.

References

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Thank you