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There is huge annual loss of both agricultural land and crops due to different reasons like construction, wearing of land, crop diseases, pest attacks, desertification, etc. United Nations Environment Program has done a recent study that showed that the food chain managed by humans is another reason for the wasting of food produced on a global level. An annual two percent increment in the amount of food produced is required to meet the basic needs of human beings in terms of hunger.
According to Abbas (1989), Asian countries like Pakistan and India are experiencing the worst condition due to greater number of inhabitants. According to APO (2002), only half the number of crops as compared to developed countries is produced in Pakistan. Sujatha et al., () says that the develpoing countries need huge scientific support for the production of more crops as a result of increasing population. Resultantly, there is number in important pulses according to different researchers (FAO, 1983; ICRISAT, 1991). an increase demand of the latest agricultural management and newest technologies in order to attain the required goals.
Mehra (1993) has found in his study that both developed and developing countries have formulated new technologies so as to enhance the quality of the crops. In this regard, micropropagation is a very promising technique. Scowcraft and Ryan (1985) has reported that tissue culture techniques have been adapted for the improvements of those plants that have huge monetary benefits. According to Mehra (1993), most new techniques in this regard are the in vitro regeneration system, development of noval hybrids through somantic hybridization and formation of transgenic plants.
Different researchers (Flick et al., 1983; Barna and Wakhlu, 1993) have shown that although different plants have increased their quality via cellular technique but still there are plants like the legumes that do not respond to the in vitro regeneration system and hence have not been improved. For these plants other new techniques need to be developed in the developing countries. In this regard, in vitro regeneration and clonal propagation need to be improved first of all as these are very important techniques.
These legume plants need that much attention because they are economically very important as food material and because they fix nitrogen present in the atmosphere and without them this process cannot be completed. Grain legumes, another name for which is pulse crop, is a vital member of family Fabaceae, which in turn occupies second rank in the line of flowering plants in nature.
Â Chickpea is the most important legume as it is on third number in the line of pulses (FAO, 1983; ICRISAT, 1991). According to Anonymous (2004), an area of 982.3 thousand ha was used for the cultivation of chickpea in 2003-04 and 611.1 thousand tons was the production with gain of 622 kg ha-1. But still chickpea yield is very less in Pakistan. There are both aboitic and biotic factors responsible for this reduction in gain like Ascochyta blight, Botrytis gray mold, dry root rot, collar rot, Fusarium wilt, pod borer, famine, salinity, and low temperature. JAYANAN et al., (2003) reported that improvements in confrontation with diseases and pests can increase the yield of this important source of proteins.
In vitro technique is very important because it has the ability, unlike the recently available technologies that are not enough, to overcome the issues of increasing world population and decreasing land availability. Direct organogenesis has been employed by different researchers for the regeneration of Cicer arietinum (Kartha et al. 1981; Islam et al. 1995; Barna, Wakhlu 1995; Anju, Chawla 2005).
Tissue Culture Studies in Legumes
Purseglove (1968) reported the presence of 690 genera and 18,000 species in family Leguminosae. After the family Gramineae, it comes on second number and out of them only 18-20 species are cultivated (Aykroyd & Doughty, 1964). According to National Academy of Sciences (1979) seeds of this family are vital food supply and are second only to cereals and this family is equivalent to high carbohydrate food like cereals and root tubers. This is of vital importance as developing countries are experiencing decrement in protein levels (Mayer, 1976).
Even though regeneration is difficult for legumes, but Nickell (1956) developed in vitro regeneration for this family. Different researchers then developed this techniques for different members of this family like mungbean (Gulati and Jaiwal 1990, 1994), peanut (McKently et al. 1989), Phaseolus vulgaris (Malik and Saxena 1992a), pea, chickpea, lentil (Malik and Saxena 1992b) and pigeonpea (Prakash et al. 1994). Different legumes were regenerated like Hinchee et al., 1988), lentil (Mahmoudian et al., 2002), blackgram (Saini et al., 2003), and pigeon pea (Thu et al., 2003) using their cotyledonary nodes as they showed the best response. Even though in vitro regeneration was used but rate of regeneration was quite low in some researches (Flick et al., 1983; Kysely et al., 1987; Prakash et al., 1994). Malik and Saxena (1992b) worked on P. vulgaris and found high amount of Benzyladenine to be very useful for shoot regeneration. Induction of multiple shoots was also found to be effected by this substance in mungbean (Gulati and Jaiwal 1994) and pigeonpea (Prakash et al. 1994).
The Chickpea (Cicer arietinum) Plant
Van der Maesen (1972) reported that chickpea (also called garbanzo, gram and Bengal gram) is grown in half dry conditions and its pollination is also self-induced. As for its name, "Cicer" is basically a Latin word that has its origin in the Greek word "Krios" from Roman family "Cicero". According to Westphal (1974) since the seed resembles the shape of a ram, so its species name i.e. Arietinum is derived from Latin word "Aries". It comes third on the line of pulse crops in the world (FAO, 1983; ICRISAT, 1991). It contains carbohydrates (48.2-67.6%), starch (41-50%), proteins (12.4-31.5%), fat (6%) other vital minerals (Ignacimuthu and Prakash, 2006).
Vegetarians give huge importance to this crop as a rich protein supply and highly grown crop. Value of its straw for animals is equivalent to other straws. Alternative agriculture that depends on nitrogen obtained from fixation also values this crop a lot as it has the capability to obtain more than 70% nitrogen through the process of symbiotic dinitrogen fixation. Kabuli-types and desi-types are the two types of cultivators of this crop.
Tissue Culture Studies in Chickpea
Somatic embryogenesis and embryo culture
Different researchers (Rao and Chopra, 1987, 1989; Riazuddine t al., 1988; Rao, 1990, 1991; Dineshkumare t al., 1994; Sonia et al., 2002) have provided different processes of regeneration including somatic embryogenesis and shoot organogenesis. Mature (Rao and Chopra, 1989) and immature leaflets (Barna and Wakhlu, 1993; Kumar et al., 1994), immature cotyledons (Eapen and George 1994, Hita et al., 1997) mature (Suhasini et al., 1994) and immature embryo axes (Sagare et al., 1993), or cell suspension cultures (Prakash et al., 1994) are the sites that have ben used for somatic embryogenesis. Krishnamurthy et al., (2000) used kanamycin and phosphinotricin for shoot regeneration of chickpea embryo axes, but these were not detailed estimates.
Different samples of chick pea like immature cotyledonary segments, immature embryo axes and mature embryo axes of cultivar PG12 were cultured and tissues were studied for somatic embryo initiation and development. Somatic embryos were cut into 10 Î¼m pieces, treated with hematoxylin-eosin and finally were seen through a microscope. Sagare et al., (1995) reported that for immature cotyledons and immature embryo axes, somatic embryos pass through a superseding callus phase for their formation, while route is direct for mature embryo axes and for young leaflets it can both be direct or indirect.
Shoot buds can also be regenerated via either direct (Shri and Davis, 1992; Kar et al., 1996; Sharma and Amla, 1998; Subhadra et al., 1998) or indirect paths (with an intermediate callus formation phase) (Khan and Ghosh, 1984; Prakash et al., 1992; Barna and Wakhlu, 1994). But only cotyledonary nodes and shoot apices have yet been used for chick pea regeneration (Sonia et al., 2002).
Jayanand et al., (2003), using cotyledonary nodes of in vitro germinated seedlings of chick pea, obtained a sample from axillary meristems and gave a very proficient and high rated way to regenerate shoots of this plant. Moreover, Rekha and Thiruvengadam (2009) used the cotyledonary nodes and axillary buds of this plant and using them gave a high frequency and replicable method of regeneration of the plantlets as well as in vitro shoot induction.
Axillary shoot proliferation (ASP)
Axillary shoot proliferation can be used for micropropagation of chickpea (1, 5, 6). Even though a true-to-type method, still the speed of this method is quite slow (11). Axillary shoot proliferation and modified single node culture were the two methods used for micropropagation of this plant. Murashige and Skoog (MS) medium enriched with 1-10 pM N6-benzyladenine and 0.01 pM a-naphthaleneacetic acid gave the maximum desired results in form of propagules. These were placed in the MS medium that has 1 pM 3-indolebutyric acid and B5 vitamins. Plants hence produced were productive and their appearance was same to those plants grown from seeds. Barna and Wakhlu (1995) showed that with greater number of plants produced per sample, MSNC is more successful regeneration method.
Using already present meristems is hopeful to lead to a more successful and rapid regeneration method. Inspite of different available methods, regenerated shoots were very limited in regard to rooting. These methods were gene-specific and for most of the time were not even replicable. Resultantly, shoot induction and in vitro rooting needs more attention.
Affect of growth regulators in organogenesis and plant regeneration
Different researchers used different materials for shoot regenaretion, enlargement, and for forming roots in ICCV-10 and Annigeri samples of chick pea. These materials were phenylacetic acid (PAA) (Ghanti et al., 2009) as well as naturally occurring auxins present in many lower and higher plant species (Abe et al. 1974, Schneider and Wightman 1986). No previous studies have yet been found in which PPA was used and direct regeneration occurred. In all the cases, an intermediate callus stage was also observed. Malik and Sexana (1992b) reported about the importance of thidiazuron in multiple shoot induction of chickpea. Benzyladenine (BA) and its role in multiple shoot induction have also been examined. Type and genetic structure of the seed sample as well as the amount of BA were the factors that affected the differentiation of shoots. This shoot differentiation was also effected by direction of the sample. Polisetty et al., (1997) found that genotype is the factor that influences what the quantity of BA should be for shoot/shoot bud regeneration. Another study found that when benzyladenine is mixed with either indolacetic acid or with naphthaleneacetic acid, then the shoot regeneration from the hypocotyls samples is highly influenced in a positive way (Islam et al., 1994). However, one drawback was that the amount of shoots obtained for each sample that was regenerated was quite low i.e. only 6-8 shoots were obtained.