Tissue Culture Studies In Pecan Biology Essay

Tissue Culture Studies In pe washbowl Biology bear witnessIn the words of Paek and Read (2007), modern biotechnology owes lots to its resolution derived from whole kit meander culture and micro times. Gottileb Haberlandt (1902) is refer red ink to as the Father of Tissue Culture, is ofttimes cited as the origin and emergence of dress wind culture and its sequent application. Plant tissue culture techniques clear become a wakeless legal document for studying and solving basic and applied problems pertaining to agriculture, industry, environment and wellness in implant biotechnology. These techniques move over great impetus in the country of propagation (Islam, 1996). Plant tissue culture is multi-dimensional field that offers ex boo thent prospects for plant emolument and knead productivity (Jain, 2001). Since the establishment of cultivation of plants, mankind is looking for for methods that aids in the mass genesis of plants employ minimum amount of m one and on ly(a)y of propagules. The ultimate result of their enquiry leads to the development of tissue culture techniques. cedarn plants having scotch meaning ar broadly speaking dispersed by bugs. Propagation of plants with and through tissue culture has become an essential and popular technique to upchuck crops that are otherwise difficult to propagate conventionally by seed and/or vegetative means. pecan is a hardwood tree species of great economic importance for its en harvestings and usually propagated through seeds. Grafting and budding are the other conventional methods of propagating pecan tree. Due to nigh(prenominal) limitations in conventional propagation methods certain relatively newer tissue culture techniques were unquestionable for tree improvements. assorted plant parts such as apical meri base, nodal explants, cotyledons or peruse explants were utilize for micropropagation of woody trees. For mul tracele lead installment cotyledonary nodal explants gat her in been employ in tree propagation (Das et al., 1996 Pradhan et al., 1998 Das et al., 1999 Purohit et al., 2002 Walia et al., 2003). Genetic variations during unfeelingness cultures and micropropagation of trees construct likewise been report (Gupta and Varshney, 1999). Some molecular markers such as RAPD and AFLP has been besides been used to detect genetic variations among in vitro clones (Gangopadhyay et al., 2003).In vitro studies for pecan improvement throughout the world are generally scanty. Tissue culture techniques arrest been developed for several tree crops, precisely previous efforts with Pecan have shown that it is difficult to propagate by in vitro methods ( woodwind, 1982). These techniques have been used in Pecan mainly for the purpose of clonal propagation. Despite the fact that it is likewise install in Northern empyreans of Pakistan (Abbotabad). So far, nonhing has been done for its developing and multiplication in Pakistan. There is to-date a s hort-fall in Pecan nut case and its products throughout the world because of the lack of rapid micropropagation methods for this tree species and disease flame during the last two decades.Various aspects of look into on Pecan includes studies on propagation (metalworker et al., 1974), seed germination and dormancy (Dimalla and Van Staden, 1977), micropropagation (Hansen and Lazarte, 1984), seed evolution and germination (Wood, 1984), somatic fertilized egggenesis (Rodriguez and Wetzstein, 1988), extrinsic trans governance (Long et al., 1995), cell suspension cultures ( fire and Wetzstein, 1997), atomic consider 25 deficiency (Smith and Cheary, 2001), launch of Zinc supply on growth and food uptake (Kim et al., 2002a), effect of nitrogen form and nutrient uptake (Kim et al., 2002b), forcing bring tips and epicormic/ recentnt buds (Preece and Read, 2003).In this section a brief review of kick the bucket is given in a manner so as to graduate(prenominal)light the curren t status of the research work in Pecan tissue culture.2.1.1 MicropropagationMicropropagation is the art and science of plant multiplication in vitro (McCown and McCown, 1999). As a concept, micropropagation was first presented to the scientific community in 1960 by Morel producing virus-free Cymbidiums. Micropropagation is a sophisticate technique for the rapid and tremendous-scale propagation of m whatsoever tree species. It has a great commercial electromotive withdraw due to extremely steep speed up of multiplication, the high plant quality and the ability to declare disease-free plants. Micropropagation has been applied to several woody tree species (Bonga and Von Aderkas, 1992). Generally, woody plants are recalcitrant to in vitro re contemporaries (McCown, 2000). The pertinency of micropropagation for woody trees has been confirmed feasible since the aspects of the sy origin have ceremonious that trees produced by this method are similar to those produced by tralatiti ous methods (Lineberger, 1980). Furthermore, Lineberger (1980) however, set forth that the major impact of plant tissue culture will not be felt in the area of micropropagation, however in the area of controlled manipulations of plants at the cellular level.Many workers have account propagation of Pecan through conventional methods (Smith et al., 1974 Brutsch et al., 1977). However these methods suffer several limitations thus pull up stakes few propagules from selected individuals (Tiwari et al., 2002). several(prenominal) efforts at Pecan tissue culture were describe by Smith (1977) and Knox (1980) but neither was successful in establishing plants in soil. However, Knox obtained few palls and plantlets when inverted nodal cuttings were used in vitro which upon transplanting did not survive. Later, Knox and Smith (1981) success richly proliferated in vitro wing-shaped worsts of Pecan using seedling explants. Success was exceptional to the organic law of hardness with onl y few strikes and root formation. Major drawbacks to clonally propagate Pecan are the poor rooting and their excerpt rate afterwards transplanting to greenhouse (Brutsch et al., 1976).In 1982, Wood successfully induce slay proliferation in axillary buds of nodal explants and report that synthetic hormones with combination of 4.0 mg/ lambert BA and 1.0 mg/ cubic decimetre IBA were most effective for shoot proliferation. Gibberellic sour (GA3) at 3.0 mg/ litre plus 0.1 mg/ litre. BA excessively enhanced shoot elongation although he was unable to subculture shoots and rooting was not achieved. In another(prenominal)(prenominal) work per organize by Hansen and Lazarte (1982) shoots were proliferated from teen Pecan in vitro and limited success was reported in terms of rooting.Hansen and Lazarte (1984) obtained single customer cuttings from 2-month-old Pecan seedlings and bring forth bud break to from sevenfold shoots on liquefiable WPM and 2 % glucose supplemented with 3.0 mg/ litre 6-Benzylamino purine (BA). The shoots developed in vitro extrinsic roots and showed vigorous root system with profuse lateral pronged from primary roots on transferring to soil after soaking in 10 mg/ litre IBA for 8 days.Corte-Olivares and co-workers (1990a) reported a performance for propagating Pecan using explants from adult trees. They collected nodal explant material during two attendant eons from grafted Western Schley trees. Specific trees representing the vegetative manikin, partially bearing phase and fully bearing phase were identified and three collections of axillary buds were made from them all(prenominal) year. Buds were cultured on Dunstan and Short (1977) basal average supplemented with 0.51 mM ascorbic demigod and 4.4 M BA. They found severe contamination problem which resulted in the info that was not amenable to statistical analysis in five of half-dozen collections of explants. Even so, in one of these five collections, shoot development an d multiplication was observe during second and third culture passages from transitional tree objet dart in four collections from teenage tree explants. Amenable data found in one or six collections where explants of all three-donor tree phase responded with shoot multiplication. The results of this preliminary study indicated that selected adult phenol types had a potential for clonally micropropagating Pecan.2.1.2 corporeal EmbryogenesisSomatic fertilized egggenesis has been known in tissue cultures of a replete(p) range of higher plants, including two angiosperms and gymnosperms (Halperin, 1995). Somatic fertilized egggenesis is a valuable tool of interest in plant biotechnology for its potential applications in clonal propagation, genetic transformation and studies involving embryo development. In step-up, somatic embryogenesis is also used for renew transgenic trees. It involves the development of somatic cells into embryos, which proceeds through a period of morphologi cal set ups that resembles zygotic embryogenesis (Dodeman et al., 1997 Dong and Dunstan, 1999). It has been reported in several moderate and tropical tree species (Gain and Gupta, 2005).It is reported that many species of tropical fruit trees could produce somatic embryos in tissue culture (Litz, 1985). In another study, equable fruit species including apple, sweetened cherry, grapes, guava etc. have also been reported to produce somatic embryos (Tisserat et al., 1979 Ammirato, 1983 Rai et al., 2007). A successful somatic embryogenesis has been reported in members of the Pecan (Carya illinoensis) family (Juglandaceae), i.e., genus Juglans nigra, Juglans hindsii using immature zygotic embryo explants (Tulecke and McGranahan, 1985). However, the application of somatic embryogenesis for the improvement of Pecan is still limited as a result of problems with showtime initiation frequencies, maintenance of embryogenic cell tilts and first base rebirth rates.Somatic embryogenesis is best known as an alternative thoroughfare to propagate Pecan via methods of tissue culture mainly due to high multiplication rates, formation of organized root and shoot axes and feasibility of mechanization. A number of studies have focused on Pecan somatic embryogenesis and renewing to complete plantlets (Merkle et al., 1987 Wetzstein et al., 1988 1989 1990 Corte-Olivares et al., 1990b and Yates and Reilly, 1990). Somatic embryogenesis has been used for induced regeneration from in vitro tissue culture, occurring in straight off from callousness, cell suspension, or protoplast culture or bringly from cells of an organized structure such as stem segment or zygotic embryo (Williams and Maheswaran, 1986). They also described the fundamental homologies amidst direct and indirect somatic embryogenesis and between single cell and multiple cell initiation. The observed conception of morphogenesis depends whether a group of cells establish and maintain duplicate behavior and influ enced by factors, which affect intercellular communication. McGranahan et al., (1987) obtained genetic transformation using somatic embryogenic cultures in Juglans. Wetzstein et al., (1996) suggested that somatic embryogenesis has the potential for propagating Pecan rootstocks and useful in introducing genes of commercial interest.Merkle et al., (1987) induced somatic embryogenesis from immature zygotic embryos of Pecan cultivars Stuart and Desirable, in spite of appearance one month following transfer from circumscribed WPM with 2.0mg/litre 2, 4-D and 0.25 mg/litre BA in the light to hormone-free medium in the dark but with low embryogenic relative frequency. Wetzstein and co-workers (1988) however, im turn out the embryogenic frequency up to 40 % for some explants sampling stages of Pecan.In another study, Wetzstein and co-workers (1989) examined the effect of cultivars, sampling date, tree ancestor of explants and duration on learn medium for the optimum yield of somatic em bryos in two cvs. (Stuart and Desirable) of Pecan. Significant variations in embryogenic reception were observed in both the cultivars. A short term exposure to 2, 4-D was shown to be quite adequate for embryogenesis in Pecan. Immature zygotic embryos collected in a developmental stage of rapid cotyledon expansion showed highest embryogenic response, i.e., 54.7 % in Desirable and 85.2 % in Stuart. No signification effect of duration on conditioning medium on embryogenic response was observed in both the cultivars. In Stuart, effect of different trees as explant stems was not significant but found significant in Desirable. However, plant regeneration and transplantation remained a limiting factor.Later, Corte-Olivares and co-workers (1990b) reported the induction of somatic embryogenesis in two cultivars (Western Schley and Wichita) with low developmental frequencies into complete plantlets. Growth regulators with different combinations had a significant effect on induction of embr yogenic callus. They proved that medium containing 2, 4-D was most effective for the induction of embryogenesis. The individual shoots isolated from shoot multiplication cultures were rooted with 49 % frequency upon culture for 4 weeks on BDS (Dunstan and Short) medium containing 14.8M IBA. Their results indicated the potential to successfully obtain complete plants from Pecan somatic embryos.Studies of Yates and Reilly (1990) on relation of cultivars response on somatic embryogenesis and ensuant plant development revealed that explants of micropylar character when removed from fruits in the smooth endosperm stage were more embryogenic than the intact ovules. strong suit containing auxin alone or auxin and cytokinins produced more somatic embryos than medium containing cytokinin alone.Furthermore, Wetzstein et al., (1990) examined make of zygotic embryo explanting time and auxin type on somatic embryogenesis during conditioning in Pecan (Carya illinoensis). Maximum embryogenesi s was observed after 15 weeks domiciliate pollination. Percent somatic embryogenesis and embryo form was significantly affected by auxin type and concentration but not the embryogenic efficiency. MS medium proved to be better than WPM for embryo germination.In another evoke study, Mathews and Wetzstein (1993) established new methods to increase plant regeneration by crying secondary embryos formation which can efficiently produce large number of clonal plants suitable for establishment in greenhouse. Silver nitrate (29.43 M) incorporation to WPM and application of 6-benzylaminopurine (100 M) on shoot apices increased maximum shoot regeneration frequency with average frequency (20 %) of plantlet conversion up to a maximum of 71 % in cv. Mahan. Later, 70 80 % of the restituted plants attained curing stage and 99 % of hardened plants were established successfully in the greenhouse.Later, Rodriguez and Wetzstein (1994) investigated callus action, embryo formation and embryo wor d structure in Pecan. Explants were cultured for one week on WPM with either NAA or 2, 4-D at a concentration of 2, 6 or 12 mg/litre and then subcultured on fresh basal medium. The best auxin intercession was 6 mg/l NAA in the induction medium, with 100 % somatic embryogenesis in cv. Stuart. Somatic embryos induced by NAA were shown to have relatively normal morphology than those induced by 2, 4-D. They reported that somatic embryo morphology affects plantlet conversion and NAA proved to be a superior auxin than 2, 4-D for the production of somatic embryos and their subsequent conversion to plants.In 1998, Rodriguez and Wetzstein critically compared morphological and histological aspects of Pecan somatic embryos induced on media with NAA or 2, 4-D. The media containing NAA or 2, 4-D has shown significant differences in the timing and pattern of initiation and development of somatic embryos. Embryos derived from callus cultures on NAA had normal morphology while those derived from cultures on 2, 4-D had higher incidences of abnormalities. Their study strongly revealed the multicelluar origin of embryos in contrast to earlier studies of somatic embryogenesis where embryos were defined as having single-cell origin (Street and Withers, 1974).Yates and Wood (1989) demonstrated organogenesis from immature embryonic axes in vitro in Pecan. Highest number of normal plants was produced from medium containing IBA, BA and kinetin at 0.5, 4.4 and 9.3 M respectively. Shoots only were produced on a medium containing cytokinins only and rooting was observed on medium with no cytokinins. In cv. Desirable greatest number of axillary shoots were elongated from embryo axes on a medium containing cytokinin only, but both with auxin and cytokinins opposite cv. Stuart.Later, Obeidy and Smith (1993), investigated organogenesis from mature Pecan cotyledons and embryonic axes. Embryonic axes at cotyledonary nodes formed 85 % microshoots and 30 % were rooted on an auxin-free medium after pre-culture in a medium with 20 M IBA. Adventitious buds emerged on callus surface previously produced on medium containing TDZ (25 M) from cotyledonary nodes and radicals.Kumar and Sharma (2005) induced somatic embryos from cotyledon explants of Walnut and Pecan. They cryopreserved these somatic embryos using non-toxic cryoprotectants, i.e., DMSO, glycerol and ethylene glycol and evaluated their choice percentage. Maximum survival percentage was observed with 5 % DMSO, 1.5 % glycerol and 3% ethylene glycol pre-treatment. In contrast, higher sucrose levels diminish survival rate and the embryos became necrotic. However, sucrose-desiccated somatic embryos pretreated with cryoprotectants survived better after one day in the liquid nitrogen.Somatic embryogenesis can be applied for efficient plant regeneration systems. It may also be utilized for introducing the genes of interest. molecular(a) markers can be used as a means of evaluating genetic stability of plants regenerated through tissue culture. Somatic embryos exhibit morphological features similar to zygotic embryos. Abnormal developments, however, frequently been observed and genetic fidelity of embryos is unknown. Therefore, the genetic fidelity of culture must be evaluated before somatic embryogenesis can be exploited. In such an interesting research work, Vendrame et al., (1999) evaluated the applicability of using AFLP analysis to assess the genetic variability in somatic embryos of Pecan (Carya illinoensis) and compared between and within embryogenic culture lines. They revealed that individual embryos derived from the same culture line exhibited high similarity and could be grouped together. However, within a culture line some embryo-to-embryo differences were also observed. They concluded that AFLP can be used as a reproducible technique to check the genetic variation among Pecan somatic embryo cultures. Larkin and Scowcroft (1981) were the first who designated variations in tissue-cultur e-derived plants as somaclonal variations. Somaclonal variations were also discover in Peach regenerates when developed from two different embryo callus cultures using RAPD (Hashmi et al., 1997). They suggested that genetic changes occurred during tissue culture. Brown et al., (1993) were also successful in genetically distinguishing among wheat suspension culture lines and also among regenerated plants through RAPD.Several studies have been reported to the use of molecular markers in at a lower placestanding the Pecan genome. The genetic diversity of Pecan populations through isozyme system has been demonstrated by Marquard 1987, 1991 Marquard, et al., 1995 Ruter et al., 2000, 2001). Conner and Wood (2001) employed RAPDs for the identification of Pecan cultivars and estimate their genetic cogitateness. The molecular evaluation of Pecan trees regenerated from somatic embryogenic cultures was carried out by Vendrame et al., (2000) using AFLPs. Grauke, et al., (2001) reported mean 2C genomic size of Pecan to be approximately 1.7 pg. Later, in another study, Grauke et al., (2003) evaluated simple sequence repeat (SSR) markers for the genetic study of Pecan. Crespel et al., (2002) stated that molecular markers are valuable in perennial crops for the construction of linkage maps. Molecular linkage maps are successfully employed in many crops for tell germplasm improvement (Pearl et al., 2004). Recently, molecular linkage maps of several tree fruit and nut crops have also been produced, including Pear (Yamamoto et al., 2002), Apricot (Lambert et al., 2004) and Walnut (Fjellstrom and Parfitt, 1994). In such another interesting work, Beedanagari et al., (2005) reported a first genetic linkage map of Pecan using RAPD and AFLP markers. These maps are an important first amount towards the detection of genes controlling horticulturally important characters such as nut size, maturity date, kernel quality and disease resistant (Conner, 1999).To initiate however work on Pecan, somatic embryogenesis has also been attempted by using cell suspension cultures. Regenerable suspension cultures established an attractive tool for the production of clonal plants and in studies involving genetic transformation. Previously, repetitive somatic embryogenesis was first reported in Pecan (Merkle et al., 1987) on solidified medium. Later, a number of research workers have improved the quantity (Wetzstein et al., 1989 Yates and Reilly, 1990) and quality (Wetzstein et al., 1990) of the somatic embryos through modified culture media and conditions. Though many improvement of the cultured media, not any previous report represented the development of somatic embryos in liquid medium. In liquid suspensions, synchronized development of the embryogenic cultures was one of the major favor over the solidified cultures.In tissue cultures of Pecan, stable embryogenic suspensions have been developed by Burns and Wetzstein (1994). They induced pre-globular stage embryo mass es on hormone-free liquid suspension cultures of Pecan to develop into somatic embryos on semi-solid medium. establish of modified solid medium (various combinations of ABA, Maltose, casein hydrolysate and filter paper overlays) treatments on somatic embryo storage reserve accumulation was investigated. Embryos analyzed for triglycerides and protein table of contents showed significant reserve deposition for some treatments but associated with undesirable deterioration in embryo morphology. The treatment that enhances the reserve accumulation was identified promoting plant recovery from suspension-derived Pecan somatic embryos.Later, in another interesting work, Burns and Wetzstein (1997) developed a method for the establishment and proliferation of developmentally stable, embryogenic Pecan suspension cultures, presenting a major improvement in embryogenic tissue culture in Juglandaceae. The established suspension cultures consisted of a mixture of pre-globular, globular stage emb ryo aggregates and freely suspended globular embryos. Their studies revealed that cultures were repetitively embryogenic and proliferated in growth-regulator-free medium. Repetitive embryogenic cultures have also been reported in Juglans regia (Tulecke and McGranahan, 1985) and Juglans nigra (Neuman et al., 1993 Preece et al., 1995), related members of the family Juglandaceae.2.1.4 Adventitious RegenerationAdventitious regeneration means the production of extrinsic shoots and buds from tissue other than axillary buds, e.g., the cotyledonary explants. The most common explants for adventitious regeneration of woody plants are cotyledons. They may either be from mature or immature seeds and leaf tissue from in vitro cultures. Although adventitious regeneration is generally undesirable for clonal micropropagation, it can represent an excellent opportunity to regenerate plants from various tissues. Also the propagation rates can be much higher than axillary shoot formation (Chun, 1993). Adventitious shoot formation can also be used for overcoming reproductive barrier caused by sterile male/ female plants (Kantia and Kothari, 2002).Conventional propagation techniques for woody fruit species are slow and possess several difficulties due to long generation cycles and high level of heterozygosity (Sriskandarajah, et al., 1994). There is a need to develop in vitro methods that could be available to speed up the breeding process for crop improvement. Many woody plant species resisted the establishment of an efficient system for regenerating plantlets due to genetically driven in vitro recalcitrance (McCown, 2000 Shing et al., 2002). However, in vitro adventitious regeneration has been achieved from various plants of several woody tree species (Maggon and Singh, 1996 Nagori and Purohit, 2004). It was reported that under identical conditions the shoot regeneration percentage varied depending on the source and type of explants used (Gentile et al., 2002 Grant and Hammatt, 2000). A higher percentage of shoot regeneration was attained from juvenile leaf explants as compared to adult leaves in Prunus dulcis (Miguel et al., 1996). Regeneration has also been achieved from the leaves of apricot (Burgos and Alburquerque, 2003), black cherry (Hammatt and Grant, 1998) and sweet cherry (Matt and Jehle, 2005). Regeneration of adventitious shoots has been reported from immature cotyledons of Peach (Yan and Zhou, 2002) and almond (Ainsley et al., 2001). In addition, regeneration using mature cotyledons has been reported for Peach (Pooler and Scorza, 1995), cosmetic cherries (Hokanson and Pooler, 2000) and sweet cherry (Canli and Tian, 2008). Regeneration through adventitious shoot formation was achieved in Feronia limonia using hypocotyls segments by Singhvi (1997).In vitro studies for Pecan improvement throughout the world are scanty. However, adventitious regeneration was reported in some members of the family Juglandaceae, e.g., Juglans nigra (Neuman et al., 1993) and Juglans regia (Chvojka and Reslova, 1987). This phenomenon may be of particular significance for extremely recalcitrant woody plant species such as Pecan also.Long et al., (1995) reported an unexpected observation that was the production of adventitious shoots from the cotyledonary explants of Juglans nigra, placed on WPM medium containing 2, 4-D and TDZ. Obeidy and Smith (1993) showed similar adventitious buds arising from callus cultures of mature Pecan (Carya illinoensis) embryonic tissues. Their shoots were regenerated from explants placed on MS medium with 25 M TDZ.Later, in the experimental work of Neuman et al., (1993), no shoot organogenesis were recorded when immature cotyledonary explants were placed on WPM medium containing 2, 4-D and TDZ. However, Preece observed shoot organogenesis in Juglans nigra (unpublished data) from cotyledonary explants placed on WPM medium containing 2, 4-D and TDZ. Adventitious shoots were readily multiplied through axillary shoot pro liferation. Biotechnology utilizing adventitious regeneration may also present a new opportunity for the improvement of woody plant species.2.1.5 fable Micropropagation MethodsPrevious tissue culture work involved micropropagation of cuttings obtained from seedlings or buds of trees big(a) under field conditions. The rooting of these shoots is slow or they may not be rooted as well. On the other hand, contamination was another major constraint encountered when these shoots are used for in vitro cultures. Shoots taken from open-air(prenominal) usually have microbes in tiny cracks of bark, not removed through disinfestations causing in vitro contamination of cultures (Preece and Read, 2003). Therefore, some other relatively newer techniques have been developed that utilizes the parts of the plants (branch tips and/ or stem segments) during dormant season and force new growths in a greenhouse environment. These techniques, such as shoot forcing as well as forcing epicormic buds may provide a breakthrough in the micropropagation of woody plants as well as for herbaceous species. These forcing techniques also have the potential for commercial propagation of plants. Research has been conducted on shoot forcing for years but much focus was on shoot tip harvested from trees and shrubs during the dormant season (Read and Yang, 1991). For heap shoot forcing, shoot tips of specific aloofness (20-25 cm long) were cut, surface disinfested and placed in a dissolving agent containing 8- hydroxyquinoline turn (8-HQC) and different growth regulators (Yang and Read, 1992, 1993). On the other hand, large branches (40 cm long) excised from juvenile portions of the trees and shrubs can also be used to force plenty shoots on a greenhouse media (Harmer, 1988 Cameron and Sani, 1994, Henry and Preece, 1997a, b). No forcing solution is used in this technique. These forced softwood shoots can be rooted as stem cuttings (Henry and Preece, 1997a). Softwood shoots can also be util ized as explants source for in vitro studies and micropropagation (Preece, 2003).Clonal propagation is achieved by culturing nodal explants taken from in vitro seedlings or form field-grown adult trees. Hence, for in vitro establishment of softwood shoots, there is a need to obtain explants with minimum of contamination. Read and Yang, (1988, 1989) disinfested the shoot tips treating with a solution of 0.78 % NaOCl containing Tween-20. Shoot tips were forced by placing in a forcing solution containing BA and GA3. They reported that the use of GA3 favored bud break and consequently increases multiple shoot production under in vitro conditions.Read and Yang (1991) later, forced softwood shoots from privet (Ligustrum vulgaris) and arrowwood (Viburnum dentatum) and tested different growth regulators in forcing solution for rooting of softwood cuttings. They reported that IBA increased number of roots per cuttings for both plants while root length increased only in Privet. On the other hand, GA3 decreased number of roots per cutting as well as bring down root length.Similarly, in another study, Read and Yang (1992) reported the influence of pre-forcing treatment on bud break and shoot elongation of lilac, Privet and Vanhoutte spiraea. Their results revealed that pre-forcing treatments increased the percent bud break by 20 % and shoots were elongated 3.0 mm greater as compared to control. However, pre-treatment effect differed with the plant species.In 1993, Yang and Read forced Vanhoutte spirea stems in forcing solution containing 8-hydroxyquinoline citrate (8-HQC), 2 % sucrose with different levels of BA and GA3 to observe their effects on in vitro cultures. They revealed that LS (Linsmaier and Skoog, 1965) medium supplemented with 5 M BAP or 5 M BAP + 1 or 5 M IAA was found to be superior for the shoot forcing in Vanhoutte spirea. BAP addition to forcing solution enhanced shoot proliferation while GA3 reduces shoot establishment in vitro.Large stem segments ha ving epicormic (dormant, latent or suppressed) buds cut during the dormant season can also be forced by placing in a suitable glasshouse medium. Large numbers of epicormic buds are present on stems of several woody tree species. Softwood shoots developed from epicormic buds on large stem segments can be used as stem cuttings in nursery industry (Cameron and Sani, 1994 Henry and Preece, 1997b).Henry and Preece, (1997a) investigated the production of softwood shoots and their subsequent rooting from maple species. The percentage of softwood shoot production varied comfortably within the species and clones of genus Acer. However, greater (59 %) number of softwood shoots was rooted in red maple as compare to either in sugar (15 %) or Japanese maple (26 %). Furthermore, Henry and Preece, (1997b) studied the influence of length and diameter of large stem segments on the production of softwood shoots from epicormic buds of selected species of genus Acer. They concluded that both stem leng th and diameter influenced the production of softwood shoots. Their study revealed that stem segments ranging from 30 40 cm long with 5.2 7.6 diameters were best for the softwood shoot production.Preece et al., (2002) developed a system for the production of softwood cuttings during the dormant season. It provides a longer exploitation season to force and root softwood segments in mid to late winter during the year of propagation for plant growth, hence, advantageous over traditional propagation methods. They suggested that intermittent mist provides the most effective forcing environment. Juvenility seems to be an important factor and it is easier to propagate plants in the juvenile growth stage than the adult phase. Similarly, microshoots originated from adult black walnut were hard to root than that of juvenile origin (Heile