November 14, 2012

1

A Report on micropropagation of Sugarcane by means of Tissue Culture

November 14, 2012

2

Abstract

Plantation white sugar factories in Bangladesh are battling with many problems in recent times. Shortage of quality raw material (i.e. sugarcane) is one of the major problems that paralyse Bangladeshi sugar industry. Investigation is necessary to determine why sugarcane as a cash crop is no longer viable to growers. It is of prime importance to quickly find a solution that satisfies both the growers and mill management. Introduction of a feature rich variety of sugarcane to the growers is a promising solution. Introduction of a sugarcane variety to growers that is disease, draught, bug resistant and obtain high yield/unit area may encourage growers but high sucrose content in sugarcane is the primary concern for mill management.

Tissue and Cell culture can play a vital role to cost effectively produce large number of disease free seed of a suitable sugarcane variety. Tissue culture is a modern process and extensive research are been being done on this novel process.

Plant tissue culture may offer certain advantages over traditional methods including: [1]

ü The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits.

ü To quickly produce mature plants. ü The production of multiples of plants in the absence of seeds or necessary pollinators to

produce seeds. ü The regeneration of whole plants from plant cells that have been genetically modified. ü The production of plants in sterile containers that allows them to be moved with greatly

reduced chances of transmitting diseases, pests, and pathogens. ü The production of plants from seeds that otherwise have very low chances of

germinating and growing, i.e.: orchids and nepenthes. ü To clean particular plants of viral and other infections and to quickly multiply these

plants as 'cleaned stock' for horticulture and agriculture.

The list above mentions certain advantages of plant tissue culture over traditional methods of propagation. So it is obvious that sugarcane growers will prefer tissue cultured seed over traditional one.

So multiplication of a sugarcane variety with desirable feature by means of tissue culture may be the first step to address the problem of insufficient supply of quality sugarcane to sugar mills.

November 14, 2012

3

Scope of research

A quick internet search indicated that sugarcane tissue culture research is carried out in different countries including India, Pakistan, The Philippines & Brazil etc. In our sub-continent India & Pakistan have some success story regarding tissue culture of sugarcane. Considering the similarities between Climate Condition of sub-continental countries Indian & Pakistani research were given priority.

Internet search also indicated that in Bangladesh very few researches have been carried out on sugarcane tissue culture. [2]

November 14, 2012

4

References

[1]http://en.wikipedia.org/wiki/Plant_tissue_culture

[2]http://www.baptcb.org/ptc/search.asp?YEAR=sugarcane&B1=Search

November 14, 2012

5

Tissue culture

Tissue culture is the cloning of plants from mother plant. Tissues from mother plant are taken out and grown in nutrient media following the protocol. A protocol for a particular variety is a detailed procedure to multiply it. Development of protocol is an iterative and research oriented process. The process involves selection of a mother plant, developing the mother plant into plantlets under controlled conditions, development of roots of these plantlets and finally hardening the plant in the greenhouse.

Tissue culture: uses

Tissue culture technology is being widely used for large-scale plant multiplication. The commercial technology is primarily based on micropropagation (growing plants from small pieces of tissue under sterile conditions in a laboratory on specially selected media), in which rapid proliferation is achieved from tiny stem cuttings, axillary buds, and to a limited extent from somatic embryos, cell clumps in suspension cultures and bioreactors.

Micropropagation of seed cane through Tissue Culture technology is useful in developing large scale production of true to type and disease-free sugarcane plantlets using apical meristem culture technique. Faster multiplication of a sugarcane variety can be done.

Tissue Culture for Sugarcane

Sugarcane is a very popular cash crop helping farmers make good profits from its cultivation. Cultivation of tissue culture sugarcane has been found to be better than the traditionally grown one with respect to uniform and faster growth, high yield and more canes, free from diseases. The tissue culture sugarcane has higher sugar recovery and sucrose content as compared to the nodal cuttings.

November 14, 2012

6

Use of Tissue Culture in Sugarcane Farming [1]

Tissue Culture technology is useful in developing plantlets of any plant species in the laboratory from any part of the plant. Using apical meristem (growing part of sugarcane) culture technique faster multiplication of a sugarcane variety can be done. Apical meristem is dissected and inoculated on a growth medium having definite nutrient composition. The apical meristem starts producing tillers in the laboratory after about 45 days of incubation in temperature and light controlled conditions. The procedure of separation of tillers, inoculation in a suitable medium and incubation under suitable conditions is repeated so that from one apical meristem one can develop millions of plantlets in a period of seven to eight months. The plantlets thus produced are absolutely disease free and true to their original variety from which apical meristem is obtained. The plantlets developed in the laboratory are hardened in temperature and humidity controlled green houses. The well hardened plantlets thus developed are useful in planting Breeder's seed nursery in three tier system of seed multiplication.

November 14, 2012

7

Features of tissue cultured sugarcane plantlets [2]

1. The plantlets are true type and disease free sugarcane plantlets.

2. 98-100% survival of the hardened plantlets under field condition.

3. If one hectare tissue cultured plantlets planted seed plot is raised by planting 15,000 plantlets per hectare planting material for planting 20 to 25 hectare is obtained in 10 months duration if 3 eye-budded setts are used for planting.

4. Quicker and more than 95% germination ability of the planting material obtained from tissue cultured plantlets

5. Planting material obtained from tissue cultured plantlets if used for commercial plantation there is increase in sugarcane yield to the tune of 28 to 30 tons per hectare.

Major Advantages of Tissue cultured Seed [3]

1. Plants are vigorous, healthy, disease free and of a premium quality.

2. Yield increases from 50 - 100 % depending upon environmental conditions.

3. Sugar recovery increase by 1 - 2 %.

4. Four ratoon crops can be achieved successfully.

5. Seed sets germination is almost 100% which reduces the seed requirement to (one third).

6. Vigorous growth tendency due to profuse root system development which normally results healthy growth and no lodging.

7. Average tillerage observed was 20 - 40 tillers per plant.

November 14, 2012

8

8. Reduce lodging which will also decrease the infestation of insects and pests.

9. Lower production cost due to high sugar and cane yield per unit area.

10. Vigorous growth and development results greater disease resistance.

11. Availability of high quality vigorous and disease free seed at affordable price round the year.

12 Every bud sprouts at least thrice.

November 14, 2012

9

Different steps for sugarcane Tissue culture [4]

Apical meristem (growing part of sugarcane) is dissected and inoculated on a growth medium having definite nutrient composition.

The apical meristem starts producing tillers in the laboratory after about 45 days of incubation in temperature and light controlled conditions.

One apical meristem can develop millions of plantlets in a period of seven to eight months.

The plantlets, well-established and hardened in plastic bags, are transplanted to field condition.

Apply 16.5 kg. of granular lindane/ha to the soil after 15 days of transplantation and irrigate the field. This helps in preventing early shoot borer infestation.

If necessary, main shoot may be removed 35-40 days after transplanting.

The major earthing up needs to be done around 90-100 days after transplanting.

A seed multiplication ratio of 1:25 (planting material for 25 hectares is obtained from one hectare seed nursery) is obtained from the seed nursery planted with tissue culture plantlets.

When the well-hardened plantlets, are used, 98 to 100 % survival is recorded under field conditions.

November 14, 2012

10

Fig.: Different Stages of sugarcane Micro-propagation

November 14, 2012

11

Fig.: Different Stages of tissue culture of sugarcane.

November 14, 2012

12

Varieties of tissue cultured sugarcane

Different commercial firm have introduced many Varieties of tissue cultured sugarcane to market. Some of the varieties are mentioned below:

Aditya Biotech Lab and Research Private Limited (ABLR) [5]

The lab is promoting commercial cultivation of the following variety of tissue culture sugarcane:

Cultivar Characters

C0 86032 It is an improved variety with sucrose content as high as18-21%. It gives about 20-25 tillers/plant.

C0 91010 It is a comparatively newer variety developed in 2000 and now gaining popularity due to high yield & sucrose content.

C094012 It is a comparatively newer variety & now gaining popularity due to high yield & sucrose content.

Sugarcane Tissue Cultured Varieties released by Agriculture Biotechnologies Pakistan (pvt.) Ltd [6]

Serial no. Variety Name 1 CP-43-33 2 CP- 77-400 3 CP 81-1435 4 ABT super 5 BF - 162 6 SPSG - 26 7 SPF - 234 8 BL - 4 9 T - 10

November 14, 2012

13

Recently released varieties of sugarcane by Sugarcane Breeding Institute, Coimbatore, India [7]

Time Period Climate zone Sub-Tropical Zone Tropical zone

1980’s Co 1148, Co 1158, Co 7717, Co 7314

1990’s Co 1148, Co 89003 Co 740, Co 62175, Co 6304, Co 7219, Co 7704, Co 7527, Co 7508, Co 7504, Co 8011, Co 8014, Co 8021, Co 8208, Co 8362, Co 8371, Co 8338, Co 85004, Co 86032, Co 85019, Co 86249, Co 97009

2000’s Co 89003, Co 98014, Co 0238, Co 0118

Co 86032, Co 99004, Co 94012, Co 2001-13, Co 2001-15

November 14, 2012

14

Sample Protocol for developing tissue cultured Sugarcane seed [8]

Material and Methods

Apical portion from the shoot of sugarcane were excised from field growing plants. As sugarcane apical meristem has covering of rolled leaves, therefore, apical meristem is wrapped deep in leaf sheaths and is naturally sterilized. Therefore, it is not necessary to disinfect them. However, to prevent any contamination from outer covering of leaves, surface sterilization was carried out as follows.

For sterilization, explant was first washed with running tap water. Then treated with house hold detergent for five minutes. This was followed by second washing with tap water to remove all the traces of detergent. The explant was then treated with 10% Sodium hypochlorite solution for 15 minutes. After discarding Sodium hypochlorite, the explants were washed three times with sterilized distilled water to remove all the traces of Sodium hypochlorite. The sterilized explants were then inoculated by proper dissecting and sizing the meristem (0.5-1.0 cm) on MS (Murashige & Skoog, 1962) medium supplemented with different concentrations of BAP either alone or in combination with Kinetin or GA3. For multiplication of induced shoots hormonal concentration was decreased and shoot multiplication was observed after 24 days of shoot induction. For In vitro rooting MS medium containing different concentrations of NAA and IBA was used either alone or in combination with each other. Sucrose 3% was used in all the media.

The pH of the medium was adjusted to 5.74 with 0.1 N solution of NaOH or HCl. MS medium was used both in solid and liquid farms. For solidification 0.7% agar was used.

In case of liquid medium autoclaved cotton was used to support the plant tissues. The medium was autoclaved at 121ºC and 15 Ibs/inch2 pressure for 15 minutes. Cultures were maintained under fluorescent light having 2500

November 14, 2012

15

lux light intensity. The incubation temperature was 26◦C ± 1◦C with 16 hour light and 8 hour dark period in every 24 hour cycle. First sub-culturing was done after five weeks and rest sub-culturing after two weeks. During each sub-culturing all dead or dis-coloured or vitrified shoots were removed. Hardening was carried out in glass house under natural light conditions.

November 14, 2012

16

November 14, 2012

17

November 14, 2012

18

Results

Shoot formation from apical meristem: The criterion of good growth for newly formed shoots from apical meristem was based on the production of broad and dark green colored leaves, healthy stems and number of small germinating buds at the base of stem.

From Table 1a it is evident that in CP 77400 best results for shoot formation were obtained in AM2 medium (MS medium containing 1.5 mg/l of BAP). In this medium all explants showed shoot proliferation response within 10 days with maximum number of 1.8 shoots per explant (Fig. 2 a). By increasing the concentration of BAP, frequency of shoot proliferation was decreased and time taken for shoot formation was also delayed, In case of BL-4, shoot formation response varied from 9 to 13.5 days. Best response was obtained in AM6 medium (BAP 0.5 mg/l + Kinetin 0.25 mg/l). All explants showed 100% shoot formation within 10 days with 1.7 shoots per explant (Table 1b, Fig. 2b).

AM1 and AM8 media also showed hundred percent shoot formation but time taken was more and the number of shoots per explants were less. All other combination did not prove good for shoot formation in BL-4 (Table 1b).

In the present investigation shoot apical meristem of different sizes ranging from 0.5-5 mm was used. As shown in Table 2, time for shoot formation was increased by decreasing the size of meristem. Maximum rate of survival was achieved when meristem of 3 mm size was used. This size exhibited 100% survival with 90% regeneration potential within 12 days of inoculation. For shoot formation both solid and liquid media were used. Best results were obtained on media solidified with Phytagel at 3.0 mg/l.

Multiple shoot formation: After 4–5 weeks of shoot growth, actively growing shoots were transferred to fresh medium in jars for further growth and

November 14, 2012

19

proliferation. Both solid and liquid media were tested. Best results for shoot multiplication were obtained in liquid medium. Proliferation of shoot started and during secondary proliferation stage, lateral shoots developed from the base of newly initiated shoot. As a result a dense mass of shoots (25-30) was developed in each culture jar (Fig. 3a and b). After 20 days these bunches were further sub-divided in bunches containing 4-5 shoots and were transferred into fresh medium in jars. In this way shoot multiplication was maintained for several passages by regular transfer to fresh medium.

The best shoot multiplication response in CP 77400 was obtained in SM1 medium i.e. MS medium containing 1.0 mg/l BAP (Fig. 4a). In this medium 29 shoots were obtained after four weeks of sub-culturing. Addition of kinetin and GAз did not show any support to shoot multiplication in CP 77400. In case of BL-4, best shoot multiplication was achieved in SM5 medium i.e., MS media containing 0.25 mg/l BAP and Kin each (Fig. 4b). From Table 3 it is evident that in both the varieties rate of shoot multiplication increased by decreasing the concentration of BAP. It was also observed that shoot multiplication response was enhanced in liquid medium while solid medium delayed shoot multiplication response.

November 14, 2012

20

References

[1]http://www.vsisugar.com/india/agriculture_divisions/tissue_culture/micropropagation-technology.htm

[2] http://www.vsisugar.com/india/products/tissue_plantlets.htm

[3]http://www.pakissan.com/english/newtech/sugarcan.shtml

[4]http://www.advanceagriculturalpractice.in/w/index.php/Sugarcane_Cultivation_Package

[5]http://www.ablr.co.in/productions2.html

[6] http://www.pakissan.com/english/newtech/sugarcan.shtml

[7] http://www.sugarcane.res.in/index.php/research/popular-varieties?start=1

[8] http://www.pakbs.org/pjbot/PDFs/40(1)/PJB40(1)139.pdf