IOLOGIA (PAKISTAN) 2010, 56 (1&2), 55-62 PK ISSN 0006 3096 Facile regeneration through adventive/somatic embryogenesis from in vitro cultured immature leaf segments of elite varieties of sugarcane (Saccharum officinarum L) SAFDAR ALI & JAVED IQQL Department of otany, GC University, Lahore (SA), School of iological Sciences, University of the Punjab, Lahore (JI) ASTRACT This research work was conducted to optimize protocol for in vitro regeneration through embryogenesis, of nine elite varieties of sugarcane. In all the nine varieties, somatic embryogenesis with and without an intervening callus phase was achieved under the influence of varying concentrations of 2,4-D ranging from 1.5 to 2.5mg -l. Embryo development was seen all over the cultured explants within four weeks of culture and the transfer of either adventive embryos or somatic embryos from embryo induction medium to embryo germination medium containing casein hydrolysate (500mgl -l ) with 2,4-D (1mgl -l ) or without growth regulators favoured embryo maturation thus resulting in plantlet regeneration within 6 weeks and all the regenerated plantlets grew normally in the greenhouse. Embryogenesis and regeneration was not significantly influenced by the variety. The technique for embryo induction involved culturing of leaf discs derived from immature leaf rolls, on medium containing 2,4-D. Plantlets were ready for hardening after 10-14 week. In a comparative study, transformation efficiency for the direct embryogenic morphogenic route was more than for indirect embryogenic morphogenic route. Due to fewer subcultures, the cost of production via direct somatic embryogenesis can be reduced per transgenic plant. This method could be useful to follow and exploit somaclonal variations as well as provide a target tissue for genetic transformation studies. Key words: adventive embryos, 2,4-D, sugarcane, transformation. INTRODUCTION Sugarcane is ideal with its high efficiency of solar energy capture, since it has both high biomass yields and is easily fractionated into highly digestible (sugar) and less digestible fibrous components (fiber or bagasse) for various uses such as fuel and fiber industry. Sugarcane, in addition to furnishing 70% of world sugar (Lakshmanan et al., 2002), is a source of natural pharmaceutical compounds such as Policosanol (Sahelian, 2004). The compelling demand of refined sugar and the use of its by-products for various purposes emphasize the need to increase production of sugarcane crop. Somatic embryogenesis is a remarkable illustration of the dictum of plant totipotency, and some sporophytic and gametophytic cells undergo embryogenesis in in vitro culture conditions and differentiate into whole plant (Kohlenbach, 1978; Willium & Maheshwaran, 1986; Dodeman et al., 1997). Sweby et al., (1994) were of the opinion that if callus was transferred after four weeks from an initiation medium containing a high level of 2,4-D (3 mgl -1 ) to a
56 S. ALI & J. IQAL IOLOGIA (PAKISTAN) maintenance medium containing a low level of 2,4-D (1 mgl -1 ), no genetic variations occur. There has been an increasing interest in the application of tissue culture techniques as an alternative means of asexual, propagation and genetic transformation of economically important plants including sugarcane. Therefore the present piece of research work was intended to devise a simple, rapid and reproducible protocol for regeneration through embryogenesis of nine elite varieties of sugarcane MATERIALS AND METHODS Plant material and explant preparation: The study was initiated with nine elite sugarcane genotypes. Healthy cane tops of sugarcane varieties viz., S96SP-302, S96SP-571, S96SP-574, HSF-240, CP72-2086, CP77-400, SPF-213, S97US- 183 and S97US-102 were kindly provided by Sugarcane Research Institute, Faisalabad, Pakistan. The cane tops collected from 9 month old cane plants were used to obtain explant to raise in vitro cultures (Ali et al, 2010). Immature leaf rolls above the first node, cut into about 1-2mm thick cross-sectional slices were used as explants (Heinz & Mee, 1969; Fitch & Moor, 1993). Culture media and embryo induction: MS (Murashige & Skoog, 1962) medium [MS salts, vitamins, myo-inositol (100 mgl -1 ) and sucrose 3%)] was used as basal culture medium with ph 5.8 and various modifications in growth regulatorsupplements such as 2,4-D (0.5-3.5 mgl -l ) alone and in combination with Kinetin (1 mgl -l ) for embryo induction for each genotype. Culture conditions: Cultures were maintained at temperature (27±2 o C) for all treatments. For embryo induction, the cultures were placed in dark as well as in continuous light conditions and during regeneration studies these cultures were exposed to 16h photoperiod with light (2000-3000 lux). Plantlet regeneration: oth types of embryos (adventive and indirect) were employed on germination/regeneration medium (MS salts, vitamins, myo-inositol (100 mgl -1 ), casein hydrolysate (500 mgl -l ), sucrose 3%) and with 2,4-D (1 mgl -l ) or without 2,4-D. Rooted plantlets were shifted to pots or 2x4 inches polythene bags to acclimatize for field plantation. RESULTS Embryogenesis response to plant growth regulators Kinetin and NAA alone and in combination were found ineffective in initial studies for somatic embryogenesis. Optimum somatic embryogenesis was observed with the treatment of 2,4-D alone in the range of 1-3 mgl -l in all the varieties under study (Table 1). The highest percentage (70) of direct somatic embryos was observed at 1.5 mgl -l 2,4-D level whereas the highest percentage (90) of indirect somatic embryos was observed at 3 mgl -l 2,4-D in most of the said genotypes. Direct somatic embryogenesis From the surface of leaf roll explants, initiation of adventive embryos was observed on MS medium with 2 mgl -l after 8-10 days under either continuous or 16 h photoperiod (Fig. 1A) as well as in dark conditions in all the nine above mentioned varieties (Fig. 1). Observation under stereomicroscope revealed the appearance of transparent bubble like proembroids, which afterwards
Vol. 56 (1&2) Facile Regeneration Through Adventive/Somatic Embryogenesis 57 transformed into embryos without forming any callus. These embryos could develop into plantlets when transferred to embryo germination medium. The direct embryo induction was exclusively dependent on 2,4-D concentration and the highest level of 2,4-D for direct somatic embryo induction was observed 2.0mgl -l but the most of the varieties such as S96SP-571, S96SP-302, CP72-2086, CP77-400, SPF-213, S97US-183 and HSF-240 reflected their best potential for direct somatic embryogenesis at 1.5 mgl -l except S96SP-574 and S97US-102 where direct somatic embryo induction occurred at 2.0 mgl -1, 2,4-D. Frequency of direct somatic embryos i.e. 70% was recorded at 1.5 mgl -1, 2,4-D for the following sugarcane varieties, S96SP-571, S96SP-302, CP72-2086, CP77-400, SPF-213 except S97US-183 and HSF-240 where the percentage of direct somatic embryos was 60 (Table 1). No response was observed below 1.0 mgl-l 2,4-D in the varieties tested except SPF-213 where direct embryos induction started at 0.5 mgl -1. Similarly above 2.5 mgl -1 2,4-D level no direct somatic embryo induction was observed except in HSF-240, where direct somatic embryo formation was observed occasionally at 3.0mgl-l 2,4-D also. Var. S97US-102 responded equally well at both the said 2,4-D levels (1.5 and 2.0 mgl -1 ) in terms of direct embryo induction percentage. Adventive embryos and E callus formation occurred simultaneously within the same cultures. The percentage of embryo formation is given in detail in Table 1. Indirect somatic embryogenesis Role of various concentrations of 2,4-D for indirect somatic embryo induction was studied in aforementioned sugarcane varieties and it was found that none of the sugarcane varieties under study responded efficiently at 0.5-1.5 mg l -l 2,4-D, but in some varieties such as S97US-102, S96SP-302 and SPF-213 it was 30% whereas in S96SP-574 and HSF-240 20% induction of indirect somatic embryos was observed at 1.5 mgl -l 2,4-D. (Table 1). Indirect embryogenesis in most of the varieties under study was observed between 80 to 90% at 2.5 mgl -1 2,4-D in S97US-183, S97US-102, S96SP-571, S96SP-302, CP72-2086, CP77-400 and HSF-240. In S96SP-574 maximum somatic embryogenesis (80%) was observed at 3.0mgl-l whereas in SPF-213 at 2.0 mgl -1 2,4-D upto 90% somatic embryos were developed. Above 3.0 mgl -1 2,4-D level no indirect embryo induction was recorded except in S97US-183 and CP72-2086 where 10 and 20% indirect embryos were observed. Response of somatic embryos to growth regulators for regeneration Under continuous or 16 h photoperiod, both adventive (direct) somatic embryos as well as indirect somatic embryos (via callus) present on the surface of compact nodular green or yellowish white E calluses germinated to form complete plantlets in 6-8 weeks. Transfer of either adventive embryos or indirect somatic embryos (Fig. 3, A) from embryo induction medium [(MS salts, vitamins, myoinositol (100 mgl -1 ) with 2,4-D (Table 1) to embryo germination medium [(MS salts, vitamins, myo-inositol (100 mgl -1 ), casein hydrolysate (500 mgl -l )] with 2,4-D (1 mgl -l ) or without growth regulators favoured embryo maturation thus resulting in plant regeneration (Fig. 3 & C). Plant regeneration potential of compact nodular yellow/yellowish white or green/greenish white E calluses was maintained up to 5 subcultures, i.e. about 24 weeks of callus age. The plantlets thus obtained grew to complete seedlings (ready for hardening process) rapidly when shifted to agar gelled plain MS medium (Fig. 3 D).
58 S. ALI & J. IQAL IOLOGIA (PAKISTAN) DISCUSSION The totipotency of plant cell can be exploited by modulating culture conditions as described by Reinert (1959). The mechanisms of totipotency, however, are not fully understood so far, and are mainly discussed in relation to the concentration and ratio of plant growth regulators (Toonen & De Vries, 1996) particularly auxins. Auxins have been implicated in a disconcertingly large number of developmental events. Their roles in embryonic polarity, organ initiation, and stem cell maintenance have all been extensively reviewed (De Smet & Jurgens 2007; owman & Floyd 2008; Vanneste & Friml 2009). In many of these cases, a general model can be discerned. First, auxin accumulates in a subset of cells, triggering a change in cell fate. This is followed by establishment of a graded distribution of auxin, often setting up an axis. Finally, this axis is used, sometimes in combination with other signals, to establish zones of cell identity. In these scenarios, auxin acts both to initiate early events and subsequently to refine the locations of specific programs. In general most of the embryo induction takes place on auxin-containing media. (Nhut et al., 2000) & in most of them the presence of auxins in the culture medium, especially the synthetic auxin 2,4-D, is the factor that determines the induction of embryo formation, as reported in many cereals such as sugarcane (Chengalrayan & Meagher, 2001), wheat (Li et al., 2003), rye (Ward & Jordan, 2001).The impact of growth regulator like auxin changes with the stage of in vitro culture, the same growth regulator has different morphogenic responses at different stages of in vitro cultures as observed in these studies that 2,4-D in higher concentration induces callus formation in immature leaf tissue, lowering the level of 2,4-D induces embryogenesis while further lowering induces regeneration in in vitro induced embryos (Falco et al., 1996; Chencalrayan & Meagher, 2001; Alam et al., 2003). The decrease in 2,4-D level from 3 & 4 mg l -l seemed to be the major factor inducing in vitro embryogenesis (Fitch & Moor 1993). The green or greenish white calli formed in light produce embryos early and leisurely and regenerate readily when shifted to regeneration medium as compared to callus, induced in dark which contain more amount of non regenerative part. The early formation of embryos and faster regeneration in this case may be due to the light sensitive degradation of auxins. The embryos germination is another important step in in vitro regeneration of plantlets. Embryos responded to germination on full as well as half strength MS medium devoid of 2,4-D, however the time duration was longer in case of full strength MS medium. The behaviour of adventive and somatic embryos towards germination was almost same with the only difference of percentage of germination where adventive embryos were observed less responsive as compared to somatic embryos. The protocol described here may be used to follow and exploit somaclonal variations as it reduced time for regeneration and higher percentage of establishment of in vitro derived plantlets at a faster rate to make in vitro regeneration system cost effective minimizing the time span for plant regeneration. This culture system is the underlying technology for efficient sugarcane transformation system as this technique has been already employed for improved plant regeneration from in vitro cultures transformed in
%age embryogenesis Vol. 56 (1&2) Facile Regeneration Through Adventive/Somatic Embryogenesis 59 sugarcane through particle bombardments (Khan et. al., 2010) and may prove to be a rapid method to provide virus free stock as well. 100 90 80 70 60 50 40 30 20 10 0 S97US-183(D) S97US-102(D) S96SP-574(D) S96SP-571(D) S96SP-302(D) 2,4-D(mg/L) 0.5 1 1.5 2 2.5 3 3.5 CP72-2086(D) CP77-400(D) SPF-213(D) HSF-240(D) Note: 1. D designates direct embryo induction, without the intervening callus phase. 2. InD designates indirect embryos appearing through the intervening callus phase. Fig., 1: Effect of 2,4-D on direct and indirect somatic embryo induction in MS modified medium containing MS salts, vitamins, myo-inositol (100 mgl-1), and sucrose 3%. A Fig., 1: Direct embryogenesis in cv. HSF-240. A) Direct and indirect embryogenesis on spindle leaf discs in continuous light after six weeks. ) Direct and indirect embryogenesis on spindle leaf discs in dark after 4 week. The arrowheads pointing the direct somatic embryos developed in both the cultures at MS3 medium (1 x).
60 S. ALI & J. IQAL IOLOGIA (PAKISTAN) A Fig. 2: A) Embryogenic callus with developed somatic embryos in cv. HSF-240. ) Close view of the somatic embryos from fig 10A, cultures of 8 week at embryo germination medium (A at 1 x and at 2.5 x). A C D Fig. 3: eginning of shoot induction after 12 week on MS medium supplemented with 2,4-D 1mg/l and CH 500mg/l HSF-240 (1 x) ) Embryo germination at the age of 15th week on MS medium basal supplemented with Vitamins. C) Plantlet growth at 20th week on the same medium (1 x) D) Seedling growth stages before hardening (1 x).
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