IN VITRO CLONAL PROPAGATION IN SUGARCANE

D. Neelamathi

Sugarcane Breeding Institute

Sugarcane is globally the main source of raw material for the production of sugar. Although many countries are sugarcane producers only six of them account for 65% of the world entire sugarcane production, Brazil being the largest one. Currently a production part of the sugarcane plantlets are produced by tissue culture. Tissue culture an important area of biotechnology can be used to improve the productivity of planting material through enhanced availability of identified planting stock with desired triats.

            Plant tissue culture is a process by which parts of plants can be grown in vitro in sterile culture in a medium containing a carbon source, minerals, growth factor and growth regulators such as hormones. Tissue culture is of great value particularly in speeding up conventional breeding and propagating procedures.

            Micropropagation is the true to tyre propagation of selected genotype or varieties using in vitro culture technique. This can be achieved by enhanced release of axillary buds ie. By multiplication through growth and proliferation of existing apical shoots excised from the parent plant. This technique provides a rapid, reliable system for production of large number of genetically uniform disease free plantlets. In 1960, Professor Georges morel of university of parise discovered ‘meristem culture’ or ‘mericloning’ a technique for vegetable multiplication. The resulting plants are called ‘mericlones’ where all plants would be identical. The explants of meristem tip culture may either be applicable meristematic dome or the apical dome with few subjacent leaves primordial. The undifferentiated growth tip, the meristem is often free from viruses that infect the rest of the plant. This is because the meristem is often free from viruses that infect the rest of the plant. This is because the meristematic cells divide and increase so rapidly that they stay a step ahead of the spreading virus whose multiplication is slower and the new plants raised from it is virus free.

            Modern sugarcane varieties are obtained through a multistage selection scheme over a period of about 8-10 years. There is a very limited quantity of seed cane available at the time of release of new variety. Conventional methods of seed cane multiplication are slow and time consuming. The conventional seed production involved a three tier system (a) treating setts will hot water or aerated steam therapy for a specific time to kill the disease and pests harboring the seed pieces (b) treating the setts in a fungicide and (c) planting in seed nurseries to raise primary seed. The primary seed is harvested at eight months age to grow a foundation seed nursery and the foundation seed in turn is used to raise the commercial seed nurseries. It is quite possible that the neew variety could enter in its degenerative cycle earlier because of the multiplication stage in the open field. Viral disease of sugarcane like GSD, RSD etc. spread systemically in plants and are seed borne. As there are no control measures by which virus infection can be eliminated from an infected plant, the only means of avoiding losses due to viral disease is to start with seed or plant, the only means of avoiding losses due to viral disease is to start with seed or plantlets which are virus free.

Micropropagation is the best methodology available for rapid multiplication and production of quality seed free of any disease and pest which ensure maximum production, potential or varieties. The schematic representation of stages of sugarcane micropropagation is given in Fig. (i). Through this technique wilt one shoot apex theoretically about 1,80,000 plants can be produced within one year which can cover about 14 hectares of nursery area. Plant production procedure essentially consists of 4 stages (Talaja, 2001).  

Stage 1: Establishment of shoot cultures

(a) Sterilization of the material

            Actively growing shoots are collected from 3-4 months old field grown healthy plants. Outer sheaths are removed by wiping the sheath with rectified spirit. Tops with growing apices are cut approximately 7-8 cm long with few whorls of leaves. The shoot bits are then washed with soap solution (surf powder) for about 5 minutes followed by several changes with water. Then thoroughly rinsed in 70 per cent ethyl alcohol for one minute. Again rinsed with sterile water 4-5 times till alcohol is completely washed away. The material is then treated in solution containing 10% sodium hypochlorite and covered immediately with aluminium foil and shaken vigorously for 20 minutes.   

 Inoculated shoot tip

(b) Inoculation

            The flask with the surface sterilized material is taken inside the laminar flow chamber. The material is washed thoroughly 3-4 times. With sterile distilled water till the chlorine smell is removed. The apical dome along with two leaf primordial is excised with help of a sterile sharp blade and immediately placed on a filter paper support immersed in the shoot apex medium (Table 1). The culture are incubated at 26° ± 1°  C and under 16 hr light (2500 lux). For survival and growth of the meristem, transfer of the meristem to fresh media is essential for a few days. After 15 days elongated shoot apices are generally transferred to bigger container for further growth and elongation.   

 Elongated shoot tip

Axillary shoot development

Stage 2: Multiplication of shoots

            After 45 to 60 days depending on the growth of the shoot apices, the material is transferred to multiplication medium (Table 1). After 45 days new shoots will arise from the axils of the developing shoots. These are separated in small groups in fresh medium.   

Multiplication of Shoots

Stage 3: Rooting and Hardening

The stage involves the induction of roots in vitro by transferring the well grown plants into rooting medium (Table 1). A group of 4-5 plants can be kept in culture tubes containing the medium. Within 15-30 days roots will be formed. Plantlets with well developed roots are planted in polybag and hardened. The mixture for planting is prepared by mixing sieved sand, silt presumed in 1:1:1 ratio.  

Rooted plants

Stage 4: Field planting

            Established plants after 45-60 day are transferred to field in normal crop season and survival rate is 85-90 per cent. A spacing of 45 cms between plants and 90 cm between rows is recommended. Canes are ready for harvest at 8 to 10 months for seed purposes.

Table1. Media composition

Advantages of Micropropagation

            In vitro micropropagation techniques are now often preferred to conventional practices of vegetative propagation because of the following advantages not only from commercial point of view but also with regard to crop improvement program.

Table2. Rate of multiplication

This system can be initially utilized to produce the breeder seed in each factory. Foundation and certificated seed can be produced from the meristem multiplied plants through vegetative cutting. Considering various benefits of this system several sugar is produced at a considerable cost. If properly implemented it will be possible to obtain improved cane yield and increased sugar production by introducing the newly identified varieties at shorter interval as and when they are released.

Although tissue culture laboratory have been established through out India during last decade producing tissue culture plants for distribution to the farmers, there is quantum of tissue culture plant would be increasing at the higher rate the quality of plants will have to be ensured for getting desired type of quality planting material. The existing tissue culture units will have to be brought under proposed quality control mechanism to ensure production of good quality and true to type plantlets and subsequent enhanced production.

References:

Jalaja, N.C. 2001. a practical manual for the sugaecane micropropagation. A Publication
from Sugarcane breeding Institute, coimbatore.  

Sreenivasan, T.V. and N.C. Jalaja. 1992. Micropropagation of sugarcane varieties for
increasing cane yield. Sugar Journal, South India Sugarcane and Sugar Technologists Association, 19(4): 61-64.

Sundara, B. and Jalaja, N.C. 1944. Effect of intra – row spacing on mericlone transplants
of Sugarcane (Saccharum officinarum). Indian Journal of Agricultural Sciences. 64(8): 546-549.

Sundara, B. 1955. Economics of using sugarcane mericlones for commercial planting. 
Co-operative sugar, 26(6): 459-461.