Dr. R.S. Sengar Kalpana Sengar and Reshu Chaudhary Sardar Vallabh Bhai Patel University of Agriculture & Technology Meerut-250110 Sugarcane is the main cash crop of India which gives 315 million tones production from 4.8 million hectare land. India has got first position in the production of sugar. In India sugarcane industry give a great employment after cotton industry. In the recent years the sugar production has increased due to the increase production of sugarcane in this country. In future if we want to supply only with our production, there will be a pressure on the production due to the increase in population and it will be impossible to increase the production of sugarcane without new techniques and it will be again problematic to make the sufficient production of sugar. Till 2020 according to increase population of the country, to achieve the target for completing the demand of sugar by using new technique there is a need to develop such kind of new species which have resistant against insects, disease and other unfavorable conditions. With tissue culture technique it is possible to develop new species of sugarcane which have high sucrose as well as high production. In our country till 2010 the demand of sugar will be 40 million tones for which it is necessary to increase the production of sugarcane. This work can be possible through tissue culture technique because this technique can provide the self employment as well as can make the business of sugarcane seed. The sugarcane production can be increased in two ways- i) by increasing the area according to sugarcane demand, which is not possible. ii) by increasing the sugarcane production per hectare with biotechnology.
Sugarcane was originally from tropical Southeast Asia. Different species likely originated in different locations with S. barberi originating in India and S. edule and S. officinarum coming from New Guinea. The thick stalk stores energy as sucrose in the sap. From this juice, sugar is extracted by evaporating the water. Crystallized sugar was reported 2500 years ago in India. Around the eight century A.D., Arabs introduced sugar to the Mediterranean and it was cultivated in Spain. It was among the early crops brought to the America by Spaniards.
Sugarcane production greatly influenced many tropical pacific islands, most particularly Hawaii and Fiji. In these islands, sugar came to dominate the economic and political landscape after the indigenous societies had been invaded by Europeans and Americas, who promoted immigration from various Asian countries for workers to tend and harvest the crop. Sugar-industry policies eventually established the ethnic make up of the island populations that now exist, profoundly affecting modern politics and society in the islands.
Brazil is a major grower of sugarcane, which is used to produce sugar and provide the ethanol used in making gasoline-ethanol blends (gasohol) for transportation fuel.
The culture experiments on sugarcane were firstly started in 1961 at Hawaii. Now a day at the many places of world such as Australia, Fiji, Taiwan, Florida, Luciana, Maryland, Philippines, Brazil, France, Shrilanka and also in India scientists are doing experiments for the genetic improvement as well as for the development in the production of sugarcane. Now a day from the three parts of sugarcane plant tissue is taken from one of them. By using the tissue, below the apical meristem or the tissue of immature inflorescence at adjustable temperature, humidity and in presence of light in vitro complete plants used to develop with the help of plant hormones.
Plant tissue culture has been promoted by Government of India since last 2 decades to enhance the production land availability of disease free, true to type, quality planting material.
The current progress of the industry is encouraging but the expected rapid growth has not taken place. The primary reason for such a slow growth is because of low awareness level amongst the people about application of this technology. Although within the country the research groups have put lots of efforts in standardizing protocols for several plant species, the benefits have not been sufficiently demonstrated to the farmers at the field level. Therefore, the technology is not so popular at the grass root level.
Currently, the focus of the companies is mainly in the floriculture sector. However, micro propagation in sugarcane is also gaining popularity.
Currently, micro propagation technology is available on the shelf for the crops which can be utilized for field test demonstration and farming.
In sugarcane the strategy for increasing are under tissue culture plants would be three fold. Initially, the nurseries of breeder seed would be prepared by the factories using plants produced by using meristems from the mother Source garden on 100 hector which will require 15 lakhs plantlets @1500 plants/hector for planting the area. The multiplication ratio being approximately 1:20, the production of sets would be around 300 lakhs sets sufficient to over an area of about 1200 hectors, under foundation stage seed production. Further the sets for this foundation stage plants would be used to produce certified stage seed sets sufficient to cover the area of 12000 hector at an expected multiplication rate of 1:10. Further the sets produced from such certified area will be distributed to farmers for growing commercial crop of sugarcane on an area of about 12000 hectors.
Instead of producing new plants, 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. However, the production capacity upgradation may be thought of after the midterm review of the program during third year.
Sugarcane yields 10-15% increase in productivity due to tissue culture plantlets. Our state occupies 5.2 lakh hectors area under sugarcane with an average productivity or 85.5 MT/ha. Quality planting that in sugarcane is also concern a matter of in sugarcane cultivation. Once sugarcane was considered relatively less affected by pests and diseases, however during past few years, a few of rumor pests earlier nave now play a major role threatening its productivity in future. There are 13 major pests reported to damage sugarcane crops heavily.
One of the major issues apart from planting material has been that of low emergence of eyebud, thereby reducing plant population and ultimately effecting production of mislabel cane. Tissue culture technique for production of good quality planting material ensures an adequate and disease free plant to get enhanced yields.
Tissue culture is the process whereby small pieces of living tissue (explants) are isolated from an organism and grown aseptically for indefinite periods on a nutrient medium. For good success in plant tissue culture, it is good to start with explant that is meristems because these cells are capable of rapid proliferation. The used explants include buds, root tips, nodal segments or germinating seeds and these are placed on suitable culture media where they grow into an undifferentiated mass known as callus.
Because the nutrient media used in the technique support the growth of microorganisms, the explants is first washed with disinfectant such as sodium hydrochloride, peroxide or mercuric chloride. Once established, the callus can be propagated indefinitely by sub-division. The nutrient media contains growth nutrient/substances and hormones i.e. auxin, cytokinin and gibberellins. The absolute amounts of hormones vary for different tissue, tissue explants from different parts of the same plant and for similar explants from different plants.
Tissue culture techniques are being exploited to enhance crop production and to aid crop improvement efforts. Faster clonal multiplication is being exploited on commercial scale for many horticultural species e.g. oil palm, menthe, roses, carnation, etc. Tissue cultured somatic tissues are now routinely being used for conservation of these species whose seeds are recalcitrant or ones which do not produce seed at all.
Embryo culture has helped in rescuing hybrid embryos enabling the recovery of many inter specific hybrids and haploid plants. Shoot tip (meristem) culture plays a vital which is of great importance in germplasm exchange, and the development of serological techniques for the detection of viruses in plant materials is a great help to the efforts in this direction.
Callus culture method (Somaclonal variation):- Plant regenerated from tissue and all cultures show heritable variation for both qualitative and quantitative traits; such a variation is known as somaclonal variation. Somaclonal variation has been described in potato, tomato etc. some variants are obtained in homozygous condition in the plants regenerated from the cells in vitro (R0 generation), but most variants are recovered in the selfed progeny of the tissue culture regenerated plants (R1 generation). Somaclonal variation most likely arises as a result of chromosome structural changes, e.g. small deletions and duplications, gene mutations, plasma gene mutations, mitotic crossing over and possibly, transposons. Somaclonal variation may be profitably utilized in crop improvement since it is reduces the time required for releasing the new variety by at least two years as compared to mutation breeding and by three years in comparison to back cross method of gene transfer.
In this method the tissue of leaf roll inside the stem or inflorescence or parenchymatic tissue is used. That plant part which is going to be used firstly under go for surface sterilization and after that in sterile atmosphere the small pieces of plants used to put in vitro in a special type of nutrient medium (Murashige and Skoog (MS) medium) on a fixed atmosphere in the dark. The semisolid medium is prepared by several kinds of minerals, vitamins, plant hormones, sucrose and agar etc. The callus developmental process starts after one week in this medium. After the development of sufficient amount of callus, this is transferred to another new nutrient medium which posses the regeneration hormone (kinetin). Now these test tubes put at 22-250C temperature in adjustable atmosphere in presence of 2000 Lux light (approximate 12 hours of a day) condition. After 10 days callus starts the regeneration process. For the multiplication of the plant it is transferred to another liquid nutrient medium after 4-5 weeks which posses a hormone (naphthalene acetic acid) necessary for the root development. In this medium after the transfer of plants the roots are completely developed in 20-25 days. After that plants put outside from test tubes and the roots are used to clean with water very well and then each and every plant is transferred to non-bacterial soil and sand in polythene bags and then these bags put inside the green house where plant get a sufficient time to maintain theirself according to the temperature and humidity of natural atmosphere when plants being maintained on favorable temperature. Only then they used to put in the field according to need. Through this method there is a possibility of variations in the sugarcane plant. At the time of callus development process there is a change in the chromosomes of sugarcane cells due to which tissues shows structural changes. Besides parental plants the plants developed from callus have both improved or nutritional variation. So the plants developed through this method found “Somaclonal variants”. The experiments at Hawaii Sugarcane Research Centre shows that the plant developed through callus have more resistance against ‘Eye spot’ diseases. In the same way some somaclonal variants show more resistance against ‘Fiji’ and ‘Downy moldue’ diseases. The experiment also proves that these somaclonal variants have not fixed resistance against disease. After 10 years vegetative propagation of these variants these species becomes disease receptive same to the parental species, some times these variants are also found due to genetic changes in tissue during the development of callus. These plants have more percentage of sugar than parental plants. Hence, there is a great possibility to develop a disease resistant and high sugar yielding plants with the help of callus culture technique. The plants developed from tissue culture shows the zero ratio of ratoon stunting disease whereas the ordinary plant shows 25-100%. In the same way the sugarcane mosaic virus ratio was plant developed from tissue culture technique the sugar percentage per tone was found 4.6% less than tissue culture plants and also in the same experiment when the data collected it was found that the crop developed from tissue culture shows 12.2% more production and 5% more sugar than ordinary crop.
Apical Bud and Meristem Culture:- Micropropagation of sugarcane seed through tissue culture using apical meristem which will go along way in fast multiplication of new promising varieties and rejuvenating and prolonging the life span of outstanding varieties under cultivation.
There is a low possibility of variation in the plants, developed from meristem culture technique then callus technique. In this method the apical bud is situated in apical meristem is used to regenerate the plant. In this method the upper surface of apical bud is sterilized from sodium hypo chloride or mercuric chloride. After that in virus less atmosphere sterile tissue in presence of nutrient chemical hormone and liquid at favorable temperature and light. In this technique nutrients can be used semisolid or in any other stage. Apical bud start development after 10-15 days of being placed on nutrient medium and after 4-5 weeks a complete plant developed. After that plants are transferred to another test tubes an a new nutrient medium where plants starts multiplication. After that each and every plant used to put separately in root developing artificial medium. After the development of roots plants transferred in polythene bags and then transferred to green house to the favorable condition. Normally as in this technique plants are developed through completely natured stabilized buds with genetic structure hence, these do not shows structural variation. But for fast multiplication when plants used to structure formed on tissues section on which several new plants developed and same the plants developed from callus these plants also different from parental plants and developed as “somaclonal variant”. Hence in using apical culture or meristem culture technique to maintain the quality of parental plants in it there is a need to put new stock culture time to time. If all the necessities for culturing are available then only by one apical bud 2,62,144 plants are easily developed in laboratory within 1 year (table-a). Hence this technique is helpful to make the great availability of seed. Normally/Generally for sugarcane planting the seed is used having 2 or 3 eyes which have the possibility of several bacteria and diseases acceptive. The plants developed from such kind of sugarcane become infected from the several kinds of diseases and insects at the time of germination by which the produced sugarcane crop as well as sucrose both is affected. In tissue culture technique only disease free part is used for developed in sterile test tubes. Hence plants rapidly developed. Due to healthy starting these plants posses more power to resist against the bacteria of soil and surroundings. That’s why these plants have the less ratio of disease and give good production as well as sugar.
Table-a- The description of plant development through a single meristem by tissue culture in one year-
Due to sugarcane cancer several species of sugarcane are laps, for eg; Co 1148, Co 1158 etc. In North India, the high yielding crop CoS-764, is going to laps due to sugarcane cancer which we can conserve by biotechnology (tissue culture). The sugarcane germination is about 35-45% in North India by which there is low yielding due to free space left in the field. Several institutes are using biotechnology for high yielding of sugarcane which is disease free and healthy. The tissue cultured plants give high yield due to disease resistant. Hence become necessary to develop a protocol of tissue culture for conservation and enhancing the high yielding sugarcane species. In laboratory the MS-medium (Table-1) with the growth regulators such as auxin, indole acetic acid or indole butyric acid (0.01-0.5-5 mg/ltr.) is prepared. The MS medium preparation method is shown in Table-2. Several stages used to develop the tissue culture plant in laboratory are as follows-
· Selection of parental plant.
· To place the parental plant tissue in test tubes.
· Germination of placed plant tissue.
· Root development in the germinated plant.
· Multiplication of sugarcane plants.
Table-1 MS medium (Murashige and Skoog, 1962)MineralsA. Main substances
D. Growth regulators: Amount differs for different species.
Table-2: The process of MS medium preparation MS medium preparation posses following stages-500 ml tissue culture grade water is taken in 1 liter beaker. The percentage of main substances is taken in this water and stirrer continuously and substances for multiplication mixed in it.
· In the same way ferrous sulphate+EDTA and vitamin is taken in this beaker.
· Sucrose and growth regulators are also mixed in this beaker.
· Now by mixing tissue culture grade water make the volume up to 1 litre.
· After that the pH of this solution adjusts at 5.7 ± 0.1 by pH meter.
· If there is a need to a agar medium then 7 gm/l agar is used and then start heating and when the solution become transparent then stop heating.
· The solution made in this way is pour 10-10 ml in test tubes and sealed by plastic cap.
· Then allow these test tubes for autoclave at 1 kg/cm2 (15 psi) for 15 minutes.
· After that cool these test tubes and put for inoculation at laminar flow.
Parental plant selection:- The selection of parental plant is a very important thing before starting the tissue culture. In it we use only those species which posses high yielding and demand such as CoS 88230, CoS 8432, CoJ-64, CoP 8421 etc. The parental plant should be disease free and healthy. The meristem & opical buds of parental plants should be choosing.
To place the parental plants tissue section in test tubes:- After collecting the buds of selected parental plants these are used to clean with water in laboratory. After that these are made fungus free by using several kinds of chemicals at laminar flow. Mainly mercuric chloride (0.5-1.5%) and sodium hypochloride (1-5%) are used and buds are placed in test tubes after drying in it which posses growth regulators with MS medium. The placed test tubes by the above manner are allowed in the culture room at a fixed temp. (25 ± 20C), optimum humidity (80%) and 8 hours light per 24 hours.
Germination of placed plant tissues:- In this stage the germination of plant tissue is occur by using different amount of growth regulators such as auxin-indole acetic acid, indole butyric acid (0.01-5 mg/l) and – cytokinin – benzyl adinine (B.A.) and kinetin (0.01-5 mg/l) (different amounts are used for different species) and in this way the plants are developed on large scale.
Root development in the germinated plants:- The several growth regulators mainly from auxin group such as indole acetic acid, indole butyric acid (0.5-5 mg/l) are used in developed plants for the roots development.
Hardening of small plants:- The hardening of small cultured plants is done in poly house or green house which posses high humidity and low temp. The per plant cost become less by using this technique. Plants take 10-12 days for hardening in poly house and green house.Applications of tissue cultured plants:-
· The seed of high yielding and healthy species can be prepared easily because 0.1-1 million plants can be developed from a single meristem in a year.
· Plants give high yield due to disease free and healthy.
· Such species do not posses any problem of germination as found in ordinary sugarcane seed.
· Being a disease free plant these are best to prepare a seed nursery in primary condition.
· The species of high yielding capacity can be conserved.