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High Frequency Differentiation of Shoots from Leaflet explants of Peanut (Arachis hypogaea L.) Sunita Daniel. N, Lakshmi Narasu, KK Sharma A simple, genotype-independent, efficient and reproducible method for plant regeneration from leaf explants of groundnut (Arachis Hypogaea L.) was developed for the production of transgenic plants. Shoot morphogenesis was induced from petiolar cut end of leaflet explants derived from 1d-old in vitro grown seedlings of groundnut genotype ICGS-44. A combination of 13.3 mM N6-benzyladenine (BA) and 5.3 mM -naphthalene acetic acid (NAA) in the MS medium with a pH of 5.8 was optimum for inducing multiple shoots. The shoot bud differentiation occurred in a polar fashion from tissues of the petiolar cut end within 2 weeks of culture in over 92% of the explants. The development of shoot buds into elongated shoots and their rooting was achieved on a hormone-free MS medium. The rooted shoots were transplanted with 100% success. The method for regeneration of whole plants developed in this study is widely applicable to different genotypes of Arachis hypogaea L. The histological events associated with shoot primordia formation in cultured leaflet explants (ICGS-44) were examined. Cytological changes were observed by day-2 culture. Mitotic activity, which was initially random, became restricted to the vascular parenchyma cells, sub-epidermal cell layers. A nodular mass of meristematic and cytoplasmically dense cells developed at the base differentiated into a meristematic zone. With in this zone shoot bud primordia and eventually multiple shoots with well developed apical meristem were formed. The short duration (15 days) and its high frequency with the time and site of differentiation make this system highly suitable for applications such as genetic transformation.
Abbreviations: SIM - Shoot induction medium Groundnut (Arachis hypogaea L.) is a popular and important food legume known for its high protein and oil content, high energy value and its adaptability to a wide range of Agro-ecological conditions (Norden, 1980). Groundnut is one of the important oil seed crop cultivated in semi-arid tropical and temperate regions. Regeneration of mature plants from undifferentiated tissues is central to the applications of biotechnology to crop improvement. An ideal regeneration system would be that the explants forms shoots with high frequency on a simple medium where time and site of differentiation are predictable. Understanding the process of differentiation from explants has become a source of agriculture innovation in plant biotechnology (Sharma and Ortiz, 2000). Several groundnut cultivars have been successfully regenerated via either organogenesis or embryogenesis (Mckently et al., 1991 Eapen and George, 1994 Sharma et al., 1993 Cheng, 1992, 1994 Livingstone et al., 1995 Chengalrayan et al., 1995 Kanyand et al., 1997 Ponsamuel et al., 1998). The present study reports that leaflet explants of groundnut (Arachis hypogaea L.) can be made to reproducibly form shoots under simple culture conditions and that the organogenic differentiation is restricted to the petiole cut end of the explants. Structural investigation contributes significantly to plant tissue culture areas such as organogenesis, knowledge of the developmental events occurring during organogenesis helps to understand the regeneration process and provides a baseline for further studies (Thorpe and Murashige, 1970). The present study reports the shoot bud differentiation and ontogeny of shoot bud from leaflet explants of groundnut.
Materials and methods Plant Material
The seeds of genotype ICGS-44, IGGC-11 and JL-24 were used in the experiments since these genotypes are widely cultivated due to their desired agronomic traits. Mature seeds of these genotypes were used as source of explants for initiating tissue culture.
Preparation of Explants
Aseptic seedlings served as the source of leaflet explants. The groundnut seeds were surface sterilized in 0.1% mercuric chloride with 1-2 drops of tween-20 for 7 min on the shaker. The bottles were returned to the laminar flow cabinet and domestos was decanted carefully. Seeds were rinsed 3-4 times with sterile distilled water and soaked in sterile water for 3 h before use. After removal of the seed coat 5 to 8 seeds were implanted in each petri-plate (90 mm x16 mm dia) containing MS basal medium (Murashige and Skoog, 1962), 3% sucrose, and 0.8% (w/v) Difco-Bacto agar for 1 d. For the preparation of leaflet explants the seed was split open and the leaflets were excised at the petiolar region of the embryo axis and cultured on shoot induction medium (SIM). At this stage the leaflets measured 2 to 3 mm (Figure 2A). The petiolar cut end of the explant was placed on culture medium with its abaxial surface in contact with the medium. The explants were cultured at a density of 8 per petri-plate and sealed with parafilm.
Culture Medium and Culture Conditions
All the experiments were conducted by using MS medium supplemented with different phytohormones that included kinetin (KN), N6-benzyladenine (BA), a-naphthalene acetic acid (NAA) and indole-3-acetic acid (IAA). The culture vessels used were (90 mm x 16 mm dia) sterile disposable petri plates for regeneration. Test tubes (150 mm x 25 mm) plugged with non-absorbent cotton were used for elongation of shoots and rooting. The medium and tubes were autoclaved at 15 psi pressure at 121 °C for 15 min. The culture medium was gelled with 0.8% (w/v) Difco-Bacto agar and pH of the medium was adjusted to 5.8 prior to autoclaving. All the cultures were maintained at 26 ± 2 °C under continuous light having 100 µEm-2S-1 irradiance provided by cool day light fluorescent lamps.
Regeneration procedure
The leaflet explants were cultured on shoot induction medium (SIM) with the petiole cut end embedded into the medium. The SIM comprised of MS basal medium containing 3% sucrose, 13.3 mM N6-benzyladenine (BA) and 5.3 mM-naphthalene acetic acid (NAA), the medium was gelled with 0.8% Difco-Bacto agar and the pH was adjusted to 5.8 prior to autoclaving. The cultures were observed over a period of 2 to 3 weeks for shoot morphogenesis from leaflet explants.
Elongation of shoots
The adventitious shoots regenerated from leaflet explants were transferred to MS basal medium for elongation in test tubes for 10 to 15 days, followed by three passages on MS medium.
Rooting
The elongated shoots were transferred to hormone free MS medium for rooting, followed by 2 passages after which the plants with well developed roots were transplanted to autoclaved sand: soil (1:1) mixture in plastic pots. The plants were covered with polythene bags and maintained at 26 ± 2 °C with about 80% relative humidity for 2 weeks prior to their transfer to 7.5 inch (dia) pots containing autoclaved field soil. The plants were maintained in the glasshouse and periodically irrigated with tap water.
Histological Procedure
To study the ontogeny of shoot bud differentiation from leaflet explants. Seeds of ICGS-44 were surface sterilized with mercuric chloride followed by three rinses with distilled water under aseptic conditions. Leaflet explants from 1d- old seedlings were excised and cultured on shoot induction medium (SIM) for different periods (1, 2, 5, 7, 10 and 14 d). Approximately 5-10 leaflets were harvested and processed at each of the above days. The explants were fixed in fixative (2 % gluteraldehyde + 2 % formaldehyde in 0.05 phosphate buffer at pH 6.8) for 24 h, dehydrated in ethanol series and 2 successive changes with n-butanol. The dehydrated specimens were infiltrated in historesin at 4oc and plastic embedding was done according to glycol methacrylate (GMA) method (O’Brien and McCully, 1981). Serial section of 3 to 5 mm thickness were cut using glass knives on a Reichert- Jung autocut microtome. The sections were stained with Toluidine Blue O (TBO) stain (Yeung and Law, 1987) for 1 min, rinsed with water, dried and observed under microscope.
Results
Shoot bud differentiation
Initially the leaflet explants from 1 d-old seedlings were cultured on medium supplemented with different concentration of BA (5.5, 13.3, 18.3 mM) and NAA (7.9, 6.3, 5.3, 1.0 mM) individually and in different combinations. In the absence of any growth regulator only roots were formed without any apparent sign of callus formation. Amongst the different media tested, MS containing 13.3 µM BA + 5.3 µM NAA (SIM) produced the highest frequency (92.3%) of multiple adventitious shoot buds. The shoot induction medium (SIM) was originally optimized for variety ICGS-44. On shoot induction medium the explants turned green, underwent considerable enlargement within two days of culture initiation. Adventitious shoots differentiated at the petiole cut end within 15 to 20 d in over 90 % of the explants (Figure 2D). Leaflets cultured on medium containing 13.3 µM BA + 5.3 µM NAA was optimum for shoot bud formation. Frequently, 4 to 6 shoots were recovered from each explant.
Orientation of the explant
General observation in leaflet culture during shoot formation was that margin of the expanded leaflet curled towards the abaxial side of the explant lifting cut ends of the base away from the medium. If the petiole cut end of the leaflet lost contact with the medium, it did not form shoots. Thus the explant with the abaxial surface in contact to the medium and base embedded on the medium is essential for shoot bud differentiation.
Age of leaflet donor seedlings
In order to study how the age of the leaflet explant effects regeneration potential, leaflets were excised from 0 to 6 d-old seedlings and cultured on MS medium supplemented with 13.3 mM BA and 5.3 mM NAA (SIM). The percentage of cultures with shoot bud differentiation decreased with the age of the seedling explant where 1 d-old leaflet showed the maximum response (Figure 2A), while those from 7 d-old seedling showed neglible regeneration. The older leaflets expanded, appeared green, produced callus, which on subsequent sub-culturing failed to regenerate shoots. Therefore, leaflet explants from 1 d-old seedlings were used in further experiments (Figure 1).
Figure 1
Shoot bud differentiation and efficacy of various cytokinins and auxins
To study the role of cytokinins and auxins on the regeneration of shoot buds the leaflet explants from mature imbibed seeds of groundnut variety ICGS-44 were cultured on MS medium supplemented with 13.3 mM BA along with different concentrations of NAA (1.0, 5.3, 6.3, 7.9 mM) or MS containing 13.3 mM BA and IAA (1.14, 2.28, 5.7 mM). The effect of BA on shoot bud differentiation was compared with two other cytokinins, viz., KN and TDZ (Figure 3). All the cytokinins induced shoot bud differentiation but with different frequencies. BA was the most effective cytokinin in terms of number of explants forming shoots and number of shoots per explant. Leaflets cultured on MS with BA and IAA turn pale yellow with in 8 d very few explants showed shoot bud differentiation. Instead, white fluffy callus was observed which on further subculture didn't show any sign of morphogenesis. Leaflets cultured on MS with varying concentrations of BA (5.5, 13.3, 18.3 µM) showed more number of shoot buds with higher BA concentration along with NAA but the shoots were of inferior quality. After two weeks, shoots arising from the petiolar cut end of the leaflets were counted, and percentage regeneration was averaged for each treatment (number of explant forming shoots / total explants cultured x 100). Combination of BA and NAA was important for obtaining successful shoot bud differentiation with high frequency (Figure 4). Amongst the different treatments tested MS containing 13.3 µM BA in combination with 5.3 µM NAA produced the highest frequency (92.3%) of multiple adventitious shoot buds that later developed into healthy shoots (Figure 2B and 2C).
Figure 3
Figure 4
Elongation of shoots
For elongation of adventitious shoots, the whole leaflet explant with differentiated shoots were transferred to test tubes containing MS hormone free medium for 1 week followed by 2 passages of 3 weeks each on MS medium for the development and elongation of adventitious shoot buds (Figure 2D). The elongated shoots were rescued at the end of each passage. The shoots were micropropagated on MS medium through nodal explants for clonal multiplication (Figure 2F).
Rooting and transplantation to the glasshouse
Regenerated shoots after elongation were transferred to hormone-free MS medium. Adventitious roots appeared on the elongated shoots within 2 weeks and developed further in 4 weeks (Figure 2G). 80 % of the rooted shoots were transferred to autoclaved sand: soil (1:10) mixture in pots and maintained in growth chamber under continuous light at 26 ± 2 °C with 80 % humidity for 2 weeks prior to transfer to glasshouse. Upon transfer of plants to the glasshouse the plants showed normal growth, matured and produced 35 to 40 pods per plant within 4 months (Figure 2H).
Genotype effect
To determine whether the in vitro shoot regeneration from leaflet explants of groundnut is genotype specific, groundnut genotypes ICGS-44, ICGS-11, JL-24 which belong to Virginia and Spanish types were tested for their organogenic response. The SIM was originally optimized for genotype ICGS-44. On SIM groundnut genotypes produced shoot buds with high frequencies (80 % to 92 %) and followed a similar pattern of growth and development (Figure 4). The regeneration system described is widely applicable to range of groundnut genotypes.
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Figure 2 (A-H) Ontogeny of shoot bud differentiation from leaflet explant of groundnut At the time of excision the leaflets measured 2 to 3 mm (Figure 2A). Those placed on the shoot induction medium began to swell by 2 d and increased in length gradually up to 10 d. After 10 d culture, the petiolar cut end of the leaflets in contact with the medium acquired pronounced nodular appearance, after 14 d culture shoots appeared at the base of the explants (Figure 2B).
Section of 0 d explants shows the epidermis, cortex and vascular elements. The cortical cells were iso diametric with vacuolated parenchyma containing one or two starch grains (Figure 5A). Section of 1 d cultured explants on SIM for 24 h showed the petiolar cut end epidermal cells entering the dividing phase. One or two starch grains in the cortical cells were observed (Figure 5B). After 2 d culture on SIM the meristematic activity in the proximity of the vascular supply had considerably increased (Figure 5C). Mitotic cell divisions with dividing nucleus could be seen (Figure 5D). In the transverse section periclinal and anticlinal divisions in the vascular parenchyma as well as in sub epidermal regions were observed (Figure 5E). Mitotic activity was localized mainly in the part of the leaflet that was near to or in direct contact with the culture medium. After 5 d culture on SIM, the explants showed the formation of lateral outgrowth and maturation of vascular elements. Mitotic activity in the vascular supply and parenchyma cells at the proximal end resulted in the formation of cytoplasmic nodular structures, which later become vascularised (Figure 5F). One notable observation was the presence of starch grains in the sub-epidermal layers. By 7 d culture differentiation of the nodular mass occurred within the cells of the meristematic zone (Figure 5H). This mass gave rise to definite meristematic zones with cytoplasmically dense cells (Figure 5G). Meristematic zones further differentiated into shoot bud initials with well organized vasculature. The meristematic cells underwent more organized development towards the periphery by 10 d culture (Figure 5I). After 14 d culture on SIM the broad meristematic zone gave rise to multiple growth centers producing shoot primordia with organized apical meristem resulting in small shoots with organized vasculature (Figure 5J). Each explant was capable of developing several meristematic nodules. This ultimately resulted in the proliferation of multiple shoot buds from a single explant (Figure 5K and 5L).
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Figure 5 (A-L)
Discussion
The classical findings of Skoog and Miller (1957) that organogenesis in tissue cultures is governed by the balance of auxin and cytokinin in the medium is demonstrated in the present study in groundnut. The hormone content of culture media is a critical factor for induction of shoots from leaflets. The influence of cytokinin in combination with low levels of auxin has been demonstrated during de novo meristem formation in other species (Franklin et al., 1991). Similar observation was made in the present study with leaflet explants, appropriate combination of BA and NAA was important for obtaining successful shoot bud differentiation with high frequency (92%). The regeneration protocol described in this report shows 3 to 4 fold increase in shoot induction from leaflet explants over that reported by Cheng et al., (1994) when they used BA (25 mg / l) and NAA (3 mg / l) Kanyand et al., (1994) with 30 mg/l thidiazuron McKently et al., (1991) with BA (40 mg/l) and Ponsamuel et al., (1998), with brassin. Mroginski et al., (1981) found that developmental age of source explant had profound effect on leaflet explants derived from young seedlings. Leaf developmental stage had been shown to affect regenerability in other species (Baker and Wetzstein, 1992). In the present study morphogenic potential is maximum at the petiole cut end of 1d-old leaflet explants. Thus, showing stage of development of the leaflet is a critical factor for induction of adventitious shoot buds. However, these results are not in agreement with those obtained in other plant species such as, tomato (Kartha et al., 1976) and Solanum lacinatum (Davis and Dale, 1979), where mature and fully developed leaves were used to regenerate plants. The reason would be that the mature leaf from groundnut have a different physiological status of development or have lost some promoting factors that were present during the early stage of development. Although shoot organogenesis has been reported from various explants in groundnut such as leaf-derived callus tissue (Mroginski et al., 1981 Pittmann et al., 1983 Seitz et al., 1987 Mckently et al., 1991 Eapen and George, 1994 Cheng et al., 1992, 1994 Kanyand et al., 1994 Chengalrayen et al., 1995 Livingstone and Birch, 1995 Baker and Wetzstein, (1992) the frequency of plant recovery is low. Chengalrayan et al. (1995) reported callusing, caulogenesis, and flowering from the base of embryo leaflets depending on hormonal composition. In most of the earlier reports, the shoots were transferred to media containing an auxin for rooting. In the present regeneration system concurrent occurrence of adventitious roots at the base of shoots on hormone-free medium was achieved, which was similar to Brassica napus (Sharma et al., 1993). Cotyledon explants have shown to be excellent explants for transformation and regeneration of fertile plants in groundnut (Sharma and Anjaiah, 2000). Leaflet explants show higher frequency (92%) of regeneration in present study.
From the histological study, it is clear that shoot buds originated from parenchyma cells associated with the phloem and the inner epidermal cells in the young leaflet. Such a developmental sequence has been observed in all systems examined histologically (Thorpe 1970 Sharma and Bhojwani, 1991 Moloney et al., 1989). In vitro conditions and exogenous supply of hormones induced the cells at the cut end to under go a different developmental pathway leading to shoot bud differentiation upon culture on shoot induction medium. Similar observation was made in Pinus radiata by Cameron and Thomson, (1969) and in Brassica juncea by (Sharma et al., 1990). The immediate channeling of the physiologically competitive explant into meristematic tissue formation, and altered morphogenetic pathway, is a key to the high shoot forming capacity of the present system. One interesting feature of the shoot forming process is the accumulation of the starch in the cells directly involved in shoot bud formation as well as those in the surrounding regions. The accumulation of the starch in the shoot-forming tissue and its utilization during shoot bud induction has been observed in many in vitro systems, since the initial observations of Thorpe and Murashige (1968) in tobacco callus (Thorpe, 1981 Brown and Thorpe, 1986 Sharma et al., 1993). From the point of view of studying development there are advantages in using direct regeneration system, due to the rapidity of morphogenesis and no requirement of subculture onto regeneration medium, de novo production of shoot primordia is extremely rapid and initially synchronous with no prolonged period of cellular differentiation. Such a regeneration system would favor easy accessibility of Agrobacterium to the meristematic cells, which are mainly surface cells during the initial co-cultivation stage of a transformation protocol (Sharma and Anjaiah, 2000). The regeneration system described in this report appears to produce multiple shoots under simple culture conditions. The system is highly reproducible, it is advantageous to regeneration and transformation due to readily available initial tissue, a short regeneration period (15-20 days), consistent regeneration, and applicable to range of groundnut genotypes.
Acknowledgements
This work was done at Genetic Transformation Laboratory, ICRISAT, India with the support and cooperation of all the staff concerned.
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