Tissue Culture Studies In Pecan Biology Essay


In the words of Paek and Read (2007), "modern biotechnology owes much to its roots derived from plant tissue culture and micropropagation". Gottileb Haberlandt (1902) is referred to as the "Father of Tissue Culture", is often cited as the origin and emergence of plant tissue culture and its subsequent application". Plant tissue culture techniques have become a fundamental tool for studying and solving basic and applied problems pertaining to agriculture, industry, environment and health in plant biotechnology. These techniques have greater impetus in the field of propagation (Islam, 1996). Plant tissue culture is multi-dimensional field that offers excellent prospects for plant improvement and crop productivity (Jain, 2001). Since the establishment of cultivation of plants, mankind is looking for methods that aids in the mass multiplication of plants using minimum quantity of propagules. The ultimate result of their enquiry leads to the development of tissue culture techniques. Woody plants having economic significance are generally propagated by seeds. Propagation of plants through tissue culture has become an essential and popular technique to reproduce 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 nut fruits and usually propagated through seeds. Grafting and budding are the other conventional methods of propagating Pecan. Due to several limitations in conventional propagation methods certain relatively newer tissue culture techniques were developed for tree improvements. Different plant parts such as apical meristem, nodal explants, cotyledons or leaf explants were used for micropropagation of woody trees. For multiple shoot induction cotyledonary nodal explants have been used 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 callus cultures and micropropagation of trees have also been reported (Gupta and Varshney, 1999). Some molecular markers such as RAPD and AFLP has been also been used to detect genetic variations among in vitro clones (Gangopadhyay et al., 2003).

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In vitro studies for Pecan improvement throughout the world are generally scanty. Tissue culture techniques have been developed for several tree crops, but previous efforts with Pecan have shown that it is difficult to propagate by in vitro methods (Wood, 1982). These techniques have been used in Pecan mainly for the purpose of clonal propagation. Despite the fact that it is also found in Northern areas of Pakistan (Abbotabad). So far, nothing has been done for its growth and multiplication in Pakistan. There is to-date a short-fall in Pecan nuts and its products throughout the world because of the lack of rapid micropropagation methods for this tree species and disease attack during the last two decades.

Various aspects of research on Pecan includes; studies on propagation (Smith et al., 1974), seed germination and dormancy (Dimalla and Van Staden, 1977), micropropagation (Hansen and Lazarte, 1984), seed maturation and germination (Wood, 1984), somatic embryogenesis (Rodriguez and Wetzstein, 1988), adventitious regeneration (Long et al., 1995), cell suspension cultures (Burns and Wetzstein, 1997), Manganese deficiency (Smith and Cheary, 2001), effect of Zinc supply on growth and nutrient uptake (Kim et al., 2002a), effect of nitrogen form and nutrient uptake (Kim et al., 2002b), forcing shoot tips and epicormic/ latent buds (Preece and Read, 2003).

In this section a brief review of work is given in a manner so as to highlight the current status of the research work in Pecan tissue culture.

2.1.1 Micropropagation

Micropropagation 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 sophisticated technique for the rapid and large-scale propagation of many tree species. It has a great commercial potential due to extremely high speed of multiplication, the high plant quality and the ability to produce 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 regeneration (McCown, 2000). The pertinency of micropropagation for woody trees has been confirmed feasible since the aspects of the system have established that trees produced by this method are similar to those produced by traditional methods (Lineberger, 1980). Furthermore, Lineberger (1980) however, described 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".

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Many workers have reported propagation of Pecan through conventional methods (Smith et al., 1974; Brutsch et al., 1977). However these methods suffer several limitations thus provide few propagules from selected individuals (Tiwari et al., 2002). Several efforts at Pecan tissue culture were reported by Smith (1977) and Knox (1980) but neither was successful in establishing plants in soil. However, Knox obtained few shoots and plantlets when inverted nodal cuttings were used in vitro which upon transplanting did not survive. Later, Knox and Smith (1981) successfully proliferated in vitro axillary shoots of Pecan using seedling explants. Success was limited to the formation of callus with only few shoots and root formation. Major drawbacks to clonally propagate Pecan are the poor rooting and their survival rate after transplanting to greenhouse (Brutsch et al., 1976).

In 1982, Wood successfully induced shoot proliferation in axillary buds of nodal explants and reported that synthetic hormones with combination of 4.0 mg/ litre BA and 1.0 mg/ litre IBA were most effective for shoot proliferation. Gibberellic Acid (GA3) at 3.0 mg/ litre plus 0.1 mg/ litre. BA also enhanced shoot elongation although he was unable to subculture shoots and rooting was not achieved. In another work performed by Hansen and Lazarte (1982) shoots were proliferated from juvenile Pecan in vitro and limited success was reported in terms of rooting.

Hansen and Lazarte (1984) obtained single node cuttings from 2-month-old Pecan seedlings and induced bud break to from multiple shoots on liquid WPM and 2 % glucose supplemented with 3.0 mg/ litre 6-Benzylamino purine (BA). The shoots developed in vitro adventitious roots and showed vigorous root system with profuse lateral branching 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 procedure for propagating Pecan using explants from adult trees. They collected nodal explant material during two consecutive seasons from grafted 'Western Schley' trees. Specific trees representing the vegetative phase, partially bearing phase and fully bearing phase were identified and three collections of axillary buds were made from them each year. Buds were cultured on Dunstan and Short (1977) basal medium supplemented with 0.51 mM ascorbic acid and 4.4 µM BA. They found severe contamination problem which resulted in the data that was not amenable to statistical analysis in five of six collections of explants. Even so, in one of these five collections, shoot development and multiplication was observed during second and third culture passages from transitional tree while in four collections from juvenile 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 Somatic Embryogenesis

Somatic embryogenesis has been known in tissue cultures of a wide range of higher plants, including both angiosperms and gymnosperms (Halperin, 1995). Somatic embryogenesis is a valuable tool of interest in plant biotechnology for its potential applications in clonal propagation, genetic transformation and studies involving embryo development. In addition, somatic embryogenesis is also used for regenerating transgenic trees. It involves the development of somatic cells into embryos, which proceeds through a sequence of morphological stages that resembles zygotic embryogenesis (Dodeman et al., 1997; Dong and Dunstan, 1999). It has been reported in several temperate 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, temperate fruit species including apple, sweet 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., 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 low initiation frequencies, maintenance of embryogenic cell lines and low conversion rates.

Somatic embryogenesis is best known as an alternative pathway 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 conversion 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 indirectly from callus, cell suspension, or protoplast culture or directly from cells of an organized structure such as stem segment or zygotic embryo (Williams and Maheswaran, 1986). They also described the fundamental homologies between direct and indirect somatic embryogenesis and between single cell and multiple cell initiation. The observed pattern of morphogenesis depends whether a group of cells establish and maintain coordinated behavior and influenced 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.

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Merkle et al., (1987) induced somatic embryogenesis from immature zygotic embryos of Pecan cultivars "Stuart" and "Desirable", within one month following transfer from modified 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 frequency. Wetzstein and co-workers (1988) however, improved 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 source of explants and duration on conditioning medium for the optimum production of somatic embryos in two cvs. ('Stuart' and 'Desirable') of Pecan. Significant variations in embryogenic response 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 sources 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 embryogenic 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 14.8µM IBA. Their results indicated the potential to successfully obtain complete plants from Pecan somatic embryos.

Studies of Yates and Reilly (1990) on relation of cultivar's response on somatic embryogenesis and subsequent plant development revealed that explants of micropylar region when removed from fruits in the liquid endosperm stage were more embryogenic than the intact ovules. Medium containing auxin alone or auxin and cytokinins produced more somatic embryos than medium containing cytokinin alone.

Furthermore, Wetzstein et al., (1990) examined effects of zygotic embryo explanting time and auxin type on somatic embryogenesis during conditioning in Pecan (Carya illinoensis). Maximum embryogenesis was observed after 15 weeks post 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 interesting study, Mathews and Wetzstein (1993) established new methods to increase plant regeneration by repetitive secondary embryos formation which can efficiently produce large number of clonal plants suitable for establishment in greenhouse. Silver nitrate (29.43 µM) incorporation to WPM and application of 6-benzylaminopurine (100 µM) 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 regenerated plants attained hardening stage and > 99 % of hardened plants were established successfully in the greenhouse.

Later, Rodriguez and Wetzstein (1994) investigated callus production, embryo formation and embryo morphology 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 treatment 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 µM 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 foe 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 µM IBA. Adventitious buds emerged on callus surface previously produced on medium containing TDZ (25 µM) 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 survival percentage. Maximum survival percentage was observed with 5 % DMSO, 1.5 % glycerol and 3% ethylene glycol pre-treatment. In contrast, higher sucrose levels decreased 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 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-culture-derived plants as somaclonal variations. Somaclonal variations were also detected 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 understanding 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 relatedness. 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 directed 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 step towards the detection of genes controlling horticulturally important characters such as nut size, maturity date, kernel quality and disease resistant (Conner, 1999).

To initiate further 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 advantage 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 masses on hormone-free liquid suspension cultures of Pecan to develop into somatic embryos on semi-solid medium. Effect 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 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 embryo 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 Regeneration

Adventitious regeneration means the production of adventitious 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), ornamental 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 µM 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 proliferation. Biotechnology utilizing adventitious regeneration may also present a new opportunity for the improvement of woody plant species.

2.1.5 Novel Micropropagation Methods

Previous tissue culture work involved micropropagation of cuttings obtained from seedlings or buds of trees grown 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 outdoor 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 softwood shoot forcing, shoot tips of specific length (20-25 cm long) were cut, surface disinfested and placed in a solution containing 8- hydroxyquinoline citrate (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 softwood 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 utilized 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 reduced 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 spirea. 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 µM BAP or 5 µM BAP + 1 or 5 µM 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 having 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 considerably 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 length 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 growing 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-Sudholt et al., 1986).

Van Sambeek et al., (2002) forced branch segments of adult hardwoods of for production of softwood cuttings from the latent buds under greenhouse conditions. A maximum of 10 - 15 numbers of visible buds were sprouted and elongated to produce softwood during February to April. They also reported sugar maple to be least productive failing to induce fewer sprouts per meter of branch wood than that of other twelve hardwood species assessed. In addition, intermittent mist throughout the day was more successful than continuous mist for forcing epicormic buds.

In 2003, Preece and Read reviewed two novel methods for micropropagation i.e., forcing softwood shoots using forcing solution and/ or forcing large stem sections in greenhouse media in many woody plants. Neither technique was used widely at that time but appeared to have great potential for woody plant micropropagation. They reported that shoot tips of 20 - 25 cm were cut and placed in a solution containing 8- hydroxyquinoline citrate (8-HQC), 2 % sucrose and different growth regulators. In order to force softwood shoots branches from juvenile tree portions were cut into sections (30 - 35 cm long) and placed horizontally in flats or benches filled with perlite. The forced soft wood shoots were excised, surface disinfested and used as explants for micropropagation. The use of intermittent mist was the best forcing environment. However, chances of microbial contamination exist when softwood shoots forced under intermittent mist and used in vitro. To minimize microbial contamination watering should be in a way that water does not contact directly with the sprouts and softwood shoots were then successfully established aseptically (Van Sambeek et al., 1997a, b).

In contrast to another research work involving silver maple (Acer saccharinum) and green ash (Fraxinus pennsylvanica), Aftab et al., (2005) reported the effect of three environments (lab, mist or fog) four media (perlite, vermiculite, 1 perlite: 1 vermiculite by volume) and H2O2 treatments on shoot forcing and subsequent transfer of explants derived from forced epicormic buds under in vitro conditions. A signification interaction was observed among perlite, vermiculite and environment with the most shoots (6.7/ stem segment) produced under mist. Explants from in vitro cultures had only 4 % microbial contamination as compared to explants from mist (92.2 %). They found that with the application of Zerotol, contamination decreased to 43 % and 46 % clean explants were established when stems were placed under mist and drenched weekly with 0.18 % H2O2. However, they demonstrated the possibility of in vitro establishment of forced softwood shoots without/ reduced microbial contamination.

Later, in another study, Preece and Read (2007) forced leafy explants and cuttings from the woody species. They demonstrated that stem diameter and stem length significantly influenced the softwood shoot production in woody species.

Previously, mostly temperate woody species have been forced from large stem sections (Preece et al., 2001; Preece et al., 2002; Van Sambeek et al., 2002). Aftab and Preece (2007) studied forcing and in vitro establishment of temperate (silver maple, green ash or Pecan) as well as for the first time in tropical tree i.e., Tectona grandis (Teak). They got 6.7 shoots per stem segments in silver maple when forced under mist on perlite/ vermiculite medium. Green ash produced 1.2 mean number of softwood shoots. In pecan, microbial contamination was the major limiting factor for softwood shoot production and establishment in vitro. However, they obtained 3 mean numbers of shoots per log when forced under lab conditions. On the other hand, in teak 5 mean number of shoots were produced under glasshouse conditions on sterilized sand.

Recently, Mansouri and Preece (2009) investigated the effect of various levels of growth regulators on softwood shoot production from large stem segments of Acer saccharinum. Softwood shoots initiated on the stems treated with 3 mM BA, produced greater number of shoots when cultured on medium supplemented with 0.01 µM TDZ. Callus formation was also observed frequently from the stem explants treated with 3 mM BA and transferred to medium containing 0.01 µM TDZ. They concluded that stem segments treated with PGR's extends the season of softwood shoot production that can be utilized as explants source in vitro.

Although the shoot forcing work was conducted on several woody tree species such as silver maple, red maple, Japanese maple, green ash and privet, but possibilities exists that it may be extended to other woody plant species such as Pecan (Carya illinoensis). However, little information regarding softwood shoot forcing in Pecan is given by the work of Aftab and Preece (2007). This technology also holds good for Pecan to produce an ample quantity of explant material that may further be utilized for propagation either in vitro or ex vitro environments.

2.1.6 Effect Of TDZ

Thidiazuron (N-phenyl-NÙŽ-1, 2, 3-thiadiazol-5-yl urea; TDZ) is a synthetic cytokinin, formerly developed by Schering and exploited as a cotton defoliant (Arndt et al., 1976). TDZ is highly stable to the plants degrading enzymes and active even at very low concentrations as compared to other synthetic cytokinins (Mok et al., 1987). Furthermore, its auxin-like (Victor et al., 1999) and cytokinins-like (Thomas and Katterman, 1986; Murch and Saxena, 2001) activity might be another possible reason for its high stability (Visser et al., 1992). Because of its magnificent ability to stimulate shoot proliferation it is selected for micropropagation over a wide range of recalcitrant woody plant species including walnut, silver maple and white ash (Huetteman and Preece, 1993). However, great care must be taken when it is employed as clonal micropropagation. Because studies have revealed that TDZ not only stimulates axillary soot proliferation but higher concentration of TDZ tends to stimulate callus formation in many woody species (Huetteman and Preece, 1993). Kaveriappa et al., (1997) reported that TDZ at higher concentrations can cause browning and explant necrosis, undesirable for morphogenic development.

TDZ has also shown to simulate shoot organogenesis from immature seeds in several woody species such as Juglans nigra (Neuman et al., 1993) and Fraxinus americana (Bates and Preece, 1990, Bates et al., 1992). On the contrary, Kulkarni et al., (2000) demonstrated that the auxin as well as cytokinins-like activities of TDZ may not permit to induce organogenesis in internodes. Huetteman and Preece (1993) observed that TDZ was most effective at lower concentrations (< 1µM) and induced greater axillary proliferation but could inhibit shoot elongation. Additionally, Gairi and Rashid, (2005) observed direct differentiation of somatic embryos on cotyledons of Azadirachta indica on low concentrations of TDZ (0.5µM). A higher concentration of TDZ (> 1µM), however, could stimulate callus formation, adventitious shoots or somatic embryos. TDZ at 10 µM regenerated adventitious shoots and somatic embryos from cotyledons of white ash (Bates et al., 1989,1992; Preece and Bates, 1990). Subsequent rooting of microshoots was unaffected or slightly inhibited by prior exposure to TDZ. Undesirable side effect of TDZ was that cultivars of some species occasionally formed fascinated shoots.

In 1995, Long and coworkers initiated somatic embryos and adventitious shoots from immature cotyledons 10-14 weeks after anthesis. They suggested that agar-solidified WPM (Woody Plant Medium) supplemented with 0.1 µM 2, 4-D and 50 µM TDZ and incubated in light for first 4 weeks is the best treatment for the induction of somatic embryos and adventitious shoots from immature cotyledonary explants. Plantlets from rooted adventitious shoots were successfully acclimatized to greenhouse conditions.

Another important research work reported successful transfer of explants derived from forced epicormic buds of Silver maple and Green ash (Aftab et al., 2005). In their work, DKW medium was supplemented with 1 µM thidiazuron (TDZ) and 1 µM IBA for axillary shoot proliferation. The results were quite satisfactory.

TDZ was also emerged as a highly a potent regulator of morphogenesis in the tissue culture of many plant species (Murthy et al., 1998). It appeared as an efficient bioregulator of morphogenesis. The recently applied approaches to study the morphogenic events initiated by TDZ have clearly revealed the details of a variety of underlying mechanism (Mok and Mok, 1982; Malik and Saxena, 1992). Some reports indicated that TDZ might act through modulation of the endogenous plant growth regulators, either directly or as a result of induced stress (Murthy et al., 1995; Hutchinson and Saxena, 1996). The other possibilities included the modification in cell membranes, energy levels, nutrient uptake, or nutrient assimilation (Chernyad'ev and Kozlovskikh, 1990; Wang et al., 1991).

Based on a review of the published literature available on TDZ, it appears that an investigation determining its role in Pecan tissue culture is lacking. This necessitates work on this aspect that may prove beneficial for Pecan improvement.