Author: Sangannavar, P A
Date published: January 1, 2012
Theoretically, tissues of shoot apical meristems are suited for plant propagation and regeneration because, these are programmed for shoot organogenesis. In practice, this method has no incidence of somaclonal variation in regenerated plants. In order to see the possibility of induction of multiple shoots from cotyledonary axiles, different experiments supplemented with growth regulators were tried. The differential effect of various concentrations of BAP on the stimulation of shoot bud formation in cotyledonary nodes have already been reported in Glycine , Pisum , Phaseolus  and Gossypium . Induction of multiple shoots in cotyledonary nodal region is useful in plants or in in vivo genetic transformation studies. We report here regeneration and induction of axillary shoots by suppressing apical dominance of shoot apex and histological studies were conducted to know the possibilities of exposing corpus layer by trimming.
MATERIALS AND METHODS
Two genotypes viz., DLSa-17 (G. arboreum) long linted desi cotton with fibre properties suitable for 40's count and SB(YF)-425 (G. barbadense) male parent of a very popular DCH-32 interspecific hybrid were used so they have potential value to use in genetic transformation studies. Seed lint was removed using H2SO4 and were washed with tap water to remove the acid completely fromthe surface. Delinted seeds were kept in 0.2% HgCl2 for 20 minutes with constant stirring followed by the repeatedwashes with sterile distilled water in laminar airflow. Seeds were allowed to germinate aseptically at 26 0C.
1. Preparation of explants
Induction of multiple shoots by removing apical dominance in in vitro condition: From 6-8 days old well developed seedlings, shoot apices were removed by using sharp blade and cultured on MS broth with different concentrations of BAP (0.1, 1.0, 2.0, 4.0, 16.0 mg/l) and TDZ (0.1, 1.0, 2.0, 4.0, 16.0 mg/l).
Induction of multiple shoots by removing apical dominance in in vivo condition: Shoot apices were removed from6-8 days old well established seedlings grown in pots by using sharp blade and applied different concentrations of BAP (2.0, 10.0, 40.0 mg/ l) and combination of NAA (0.1mg/l) and BAP (2.0, 10.0, 40.0 mg/l) by cotton swab.
Trimming shoot apices to expose corpus layer and regeneration: The shoot apices isolated from aseptically grown seedling were trimmed to remove leaf primordia, 廠h of 1mmof shoot apex, 1/3rd portion of shoot apex (Fig. 1) followed by culturing on basal MS media.
Statistical procedures and analysis: The data was analyzed under completely randomized design using MS Excel programme.
2. Histological analysis
Induction of multiple shoots by removing apical dominance in in vitro condition: From the seeding incubated after the removal of apical meristems, suitable tissues containing axillary region of cotyledons were fixed.
Trimming shoot apices to expose corpus layer and regeneration: Shoot apices isolated from aseptically germinated seeds with different types of trimming viz., removal of leaf primordia, 廠h portion of 1mm of shoot apex, 1/3 rd portion of shoot apex were fixed.
Tissue samples were fixed in FAA for 3 hours at room temperature. Fixed materials were dehydrated in a graded ethanol series and embedded in paraffin wax. Wax blocks were stored at 4 0C until further processing. Sections of 10 mm thick were cut on a microtome, affixed to microscope slides, dewaxed in Histoclear, rehydrated through ethanol series and stained with Saffranin-Fast Green. These were mounted with a xylene-based mountant DPX after dehydration through ethanol and Histoclear. Microscope slides were examined under bright field with an Olympus light microscope and images were recorded using a digital camera attached to an Olympus imaging system.
RESULTS AND DISCUSSION
In the present study possibilities of induction of shoots from the nodes of cotyledons was tried. Induction of advantageous shoots from the nodes of cotyledons may be useful in in vitro and in vivo genetic transformation studies.
Induction of multiple shoots by removing apical dominance in in vitro condition: The number of explants responded for induction of shoots fromaxiles of cotyledon of DLSa-17 was highest in MS media which is significantly higher (88%) than different media supplemented with BAP at different concentration viz 0.1 mg/l (78%), 1 mg/l (76%), 2 mg/l (80%), 4mg/l (66%) and 16mg/l (50%). Similarly when TDZ was used the per cent response in term of number of explant was also significantly lower in different concentration 0.1 mg/l (82%), 1 mg/l (76%), 2 mg/l (82%), 4 mg/l (66%) and 16 mg/l (44%) than MS (88%). The data is presented in table 1.
Similarly in SB(YF)-425, the number of explants responded for induction of shoots from axiles of cotyledon was significantly higher inMS media (66%) than different media supplemented with BAP at different concentration viz 0.1 mg/l (62%), 1 mg/l (58%), 2 mg/l (64%), 4 mg/l (52%), 16 mg/l (64%). Similarly when TDZ was used the per cent response in term of number of explant was significantly lower in different concentration 0.1 mg/l (64%), 1 mg/l (56%), 2 mg/l (58), 4 mg /l (54%), 16 mg/l (50.0%) than MS (66%).The data is presented in table 2.
In in vitro condition only two shoots one each from the axiles of cotyledon were observed. However, no advantageous shoots induction was noticed from the axiles of cotyledon in any of the media tried. In fact normal emergence of shoots was clearly evident in MS media. The emergence of cotyledonary axile shoots in the BAP and TDZ supplemented media was hindered/stunted, even after replacing these cultures with basalMS media, there is no emergence of multiple shoot. The induction of shoots from the axiles of cotyledon was evident only when apical dominance was prevented by removing SAM (Fig. 2). None of the media induced shoots from the axiles of nodes in the explants intact with apical dominance.
Induction of multiple shoots by removing apical dominance in in vivo condition: In DLSa-17, significantly highest (96%) number of plants were recorded for the induction of cotyledonary axile shoots in control (when there was no application of PGRs) followed by application 2.0 mg/l BAP (88%) and 2.0 mg/l BAP plus 0.01 mg/l NAA (80%). The lowest number of plants showing the induction of cotyledonary axile shoot was noticed in application 40.0 mg/l BAP plus 0.01 mg/l NAA(4%) (Table 3).
Similarly, in SB(YF)-425 significantly highest (92%) number of plants were recorded for the induction of cotyledonary axile shoots in control (when there was no application of PGRs) followed by application of 2.0 mg/l BAP (64%) and 2.0 mg/l BAP plus 0.01 mg/l NAA (44%). The lowest number of plants showing the induction of cotyledonary axile shoot was noticed in application of 40.0 mg/l BAP plus 0.01 mg/l NAA (8%) (Table 3).
Irrespective of exogenous growth regulator's application, the shoot induction and growth was observed onlywhen apical dominancewas suppressed by removing shoot apices. Growth regulators application to axiles of cotyledon did not result in multiple shoot induction in well established seedlings (Fig 3). Higher numbers of axile shoots were observed in control as compared to PGRs applied once, and also regeneration was affected by application of higher dose of PGRs. In cotton , Soybean  and Sorghum  did not get any multiple shoots when tried to use different combinations of PGRs for induction of multiple shoots. In our study irrespective of genotypes there was no growth regulator helps in inducing multiple shoot from cotyledon axiles of in vitro and in vivo condition.
Trimming shoot apices to expose corpus layer and regeneration: In DLSa-17, significantly higher survivability and regeneration (92.5%) was observed in control than trimmed tissues (removed primordial leaves (10%), 廠h portion of 1 mm SAM (2%) and 1/3rd cut of SAM (8%)). Similarly, in SBYF-425, significantly higher survivability and regeneration (85%) was observed in control (no trimming) than trimmed tissues (removed primordial leaves (12%), 廠h portion of 1 mm SAM (2%) and 1/3 rd cut of SAM (12%)). The data is presented in table 4.
It was observed that shoot apical meristem (SAM) of plants generates the whole green part of the plant body including the flowers. Thus it is very important tissue in the developmental biology of plants. Theoretically, the advantage of the shoot apex explant over other regeneration systems is that, plants may be obtained fromany genotype rather than from only those that regenerate from callus culture.
Themeristemis tiny structure and it is often confused with complete shoot apex which also contains leaf primordia and young leaves. Themeristemas a tissue may represent a complete pattern of cells. Each of these cellsmay differ physiologically due to its unique position in the meristem [9,10]. Cell division pattern within the apical meristem of angiosperms is highly conserved leading to the classical definition of outer tunica layer (in which cell division is restricted to an anticlinal orientation) surrounding an inner corpus (in which cell division orientation is more common) [11,12]. Despite this conservation of structure, the functional significance of tunica/corpus organization remains unclear. In several studies shoot apices were used to develop genotype independent genetic transformation studies [13-17]. For heritable gene transformation transfer of foreign gene to corpus cells is must so exposing them followed by direct regeneration from these cells is effective in developing genetic transformation protocol. So in the present study, shoot apices containing SAM were trimmed and tried for regeneration. Histological investigation indicated there was induction of shoots from the exiles of cotyledons of explants (fig 4).
Histological investigation indicated the kind of trimming followed in the present study appear to damage the integrity of the SAM resulting in no regeneration (Fig. 5). The presence of tunica layer and primordial leaf can be seen clearly in the controlled tissue while absence of primordial leaves and tunica layer was clearly observed when 廠h of 1 mm tissue from the tip of the explant removed. It clearly indicates that just removing tunica layer also affects the regenerability of the SAM, as evident in the present study which shows just 2 per cent regeneration as against the control.
The regions below the meristems are the sites of active growth, a new shoot and root tissue rapidly grow. The SAM plays a role in organogenesis, the formation of newleaves and axillary buds in a precise spatial pattern. Partial removal of apical meristem followed by colonization of Agrobacterium may increases the transformant cells in meristematic region and formation of axillary shoots directly from those transformed cells of meristematic region avoids more number of chimeras and increases the frequency of transformants.
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SANGANNAVAR, P.A.,HEGDE, P.M., CHOUDKI, V.M., SAVITA, S. G., VANTI, G. L.,
BARKEER, S.,ABDULNAYEEM., VAMADEVAIAH, H.M., KHADI, B.M.AND
KATAGERI, I. S.
Department ofGenetics and Plant Breeding,Agriculture CollegeDharwad, University ofAgricultural Sciences,
Dharwad -580005, Karnataka. E.mail: email@example.com
Received: January 16, 2012; Revised: January 26, 2012:Accepted: February 11, 2012