Morphogenetic pathway in petiole derived callus of Amorphophallus albus in vitro

Two different morphogenetic pathways, adventitious bud and corm-like structure (CLS), were observed on organogenic calli derived from the petioles of Amorphophallus albus in vitro. The organogenic calli was established via culture of petiole segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.0 mg l⁻¹ 6-benzyladenine (BA) and subculture of the petiole-derived calli on MS medium with 0.5 mg l⁻¹ NAA and 0.5 mg l⁻¹ BA. These organogenic calli were used to induce morphogenesis via culture on MS medium with various concentrations of NAA and BA. BA alone favoured adventitious bud differentiation (57.0 ± 8.3% at maximum) from the organogenic calli but inhibited CLS formation. In the presence of NAA and BA, both adventitious bud and CLS were observed in a same culture system. The maximum CLS formation (71.2 ± 9.3%) were found on MS medium with 0.5 mg l⁻¹ NAA and 2.0 mg l⁻¹ BA, associated with 26.7 ± 8.6% adventitious bud differentiation. A small part of the adventitious buds developed into normal shoots which needed rooting culture phase to form complete plants. About 80% survival rate was obtained with these plants after transplantation to soil. More than 90% of the CLSs produced complete plants with shoots and root systems, regardless of the rooting media tested. Transplantation of the CLS-derived plants to soil gave 100% survival rate. Histological observations revealed both the two morphogenetic events originated from the meristematic cells located in superficial layers of callus tissue.     

In vitro root induction in axillary microshoots of Quercus robur L.

Whilst considerable efforts have been made to optimise shoot multiplication and rooting in oak, little attention has been paid to the impact of conditions used for multiplication on subsequent root formation. An optimised technique for rooting of oak microshoots has been developed to assess the effect of cytokinin treatments applied to shoot multiplication cultures on the subsequent rooting of microshoots. We found IBA to be more effective at inducing root formation in microshoots than NAA. Efficient rooting of oak microshoots (80%) was achieved after 35 days on medium supplemented with 1.0 mg litre^-1 IBA. Lower concentrations of IBA reduced the frequency of root formation and significantly increased the time taken for microshoots to form roots. High concentrations of IBA (3.0 mg litre^-1) produced similar rooting frequencies but with significantly increased numbers of roots formed by each microshoot. However, high concentrations of IBA stimulated the production of basal callus. Rooting of microshoots was unaffected by the concentration of BA used during shoot multiplication, although basal callusing was greater in microshoots taken from multiplication medium supplemented with the highest concentration of BA (1.0 mg litre^-1) and rooted on medium supplemented with 3.0 mg litre IBA. Reducing the period of exposure to auxin to 7 days by transferring microshoots to auxin-free medium increased the frequency of root formation (84%), led to more rapid root formation and a reduction in basal callus formation.     

Influence of selected carbohydrates on rhizogenesis of shoots saucer magnolia in vitro

Nodal segements were taken from juvenile shoots of mature 100 year-old trees of saucer magnolia (Magnolia x soulangiana Soul.-Bod.) and cultured on Standardi and Catalano medium supplemented with 1.33 μmol·dm⁻³ BA, 0.54 μmol·dm⁻³ NAA, 58 μmol·dm⁻³ sucrose and 6.0 g·l⁻¹ agar-agar. After 8 weeks, separated shoots were transferred to rooting medium with half-strength macronutrients (basal medium) supplemented with 0.3% activated charcoal and one of carbohydrates: arabinose, cellulose, fructose, galactose, glucose, lactose, mannose, rhamnose, ribose, sorbose, sucrose or xylose at 20 g·dm⁻³ and 7.0 g·dm⁻³ agar-agar. After 13 weeks of culture, shoot number, fresh and dry weight of shoots and roots, total root length and number of roots/per shoot were recorded. Percentages of rooted shoots were calculated. Fructose, mannose and xylose were the most effective carbon source on shoot proliferation followed by sucrose. The rooting response was induced by cellulose and xylose. Arabinose, rhamnose and sorbose inhibited root formation. The number of adventitious roots produced per shoot was stimulated by cellulose and xylose. Total biomass (shoot plus roots) of the plantlets was the highest at fructose and cellulose.     

Establishment and in vitro regeneration studies of the potential oil crop species Camelina sativa

Camelina sativa was successfully established in vitro and systems for the regeneration of shoots from leaf explants developed. Methods for the surface-sterilisation of seeds were used which gave 95% germination, though the in vitro grown seedlings failed to develop beyond 28 days culture. In a micropropagation system, the rooting response of nodal explants was increased from a control level of 26.4% to 46.7% by the addition of 5.4 μM NAA. Leaf explants were more efficient for the regeneration of root and shoots than hypocotyls. For regeneration from leaf tissue the use of auxin (NAA) alone in the medium above a level of 0.54 μM resulted in root or callus growth. Cytokinin, in the form of BA alone failed to induce regeneration, but a combination of 4.44 μM BA and 0.54 μM NAA induced shoot regeneration at rates over 10.0 shoots per explant. Regenerated shoots were successfully transplanted to soil and flowered and set seed normally. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro clonal propagation of Nyctanthes arbor-tristis

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0–1.5 mg dm⁻³ 6-benzylaminopurine (BA), 50 mg dm⁻³ adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm⁻³ BA, 50 mg dm⁻³ Ads and 0.1 mg dm⁻³ IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm⁻³ IBA and 0.1 mg dm⁻³ IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

Auxin stability and accumulation during in vitro shoot morphogenesis influences subsequent root induction and development in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

Recent results showed that after 16 months in the field, micropropagated eucalyptus plants have an inferior root system to cuttings. Such differences may be due to the plant growth regulators supplied during the culture stages of standard protocols, which are targeted at optimising plantlet yields and not root quality. This study investigated such a proposal, focusing on auxins in an easy-to-root clone. Initial results showed that the auxin provided in the standard protocol (NAA for multiplication and IBA for elongation) enabled 100% rooting in auxin-free medium, where rooting was faster than on IBA-rooting media. When auxin supply was omitted from multiplication and restricted to NAA or IAA during elongation, rooting in an auxin-free medium was reduced to 68 and 31%, respectively, reflecting the stabilities of these auxins in plant tissues. Additionally, 15% of shoots from the NAA-medium and 65% from the IAA-medium produced roots with altered graviperception. GC–MS analysis of these shoots revealed a relationship between free IAA-availability and altered graviperception. This was further tested by adding the IAA-specific transport inhibitor 2,3,5-triiodobenzoic acid to rooting media with IBA, IAA or NAA, which resulted in 100, 70.9 and 20.6% rooting, respectively. At least 40% of the sampled root tips had atypical starch grain deposition and abnormal graviperception. It is proposed that, at least in this clone, while IBA and NAA can be used for in vitro root induction, IAA is necessary for development of graviresponse.     

Micropropagation of<Emphasis Type="Italic">Schizandra chinensis</Emphasis> BAILLON using glucose from cotyledonary nodes

A protocol for the micropropagation ofSchizandra chinensis has been developed using regenerated shoots from axillary bud explants. In preparing to do so, we found that seed type (i.e., mature vs. pre-mature) significantly influenced the rate of germination. The Woody Plant (WP) medium proved to be superior to the Murashige and Skoog (MS) medium for germination purposes. Multiple shoots were induced from cotyledonary nodes of axenic seedlings on WP media containing 6-benzylaminopurine (BA) alone or in combination with 1-napthaleneacetic acid (NAA). High frequencies of shoot proliferation and the greatest number of shoots per explant (11.6) were observed with the use of 1.0 mg L^-1 BA. We also established a culture method for proliferating shoots by repeatedly subculturing the original cotyledonary nodes on a shoot multiplication medium each time newly formed shoots were harvested. To induce root formation, glucose was supplied as a carbon-source substitution for sucrose. The best rooting rate was obtained from a WP medium supplemented with 3% glucose and 0.5 mg L^-1 NAA. Following transplantation in the field, 82% of the plantlets survived.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

In vitro culture of Safflower L. cv. Bhima: initiation,growth optimization and organogenesis

Callus induction and in vitro plantlet regeneration systems for safflower (Carthamus tinctorius L.) cv. Bhima using root, hypocotyl, cotyledon and leaf explants were optimized by studying the influence on organogenesis of seedling age, media factors, growth regulators and excision orientation. Supplementation of the medium with an auxin: cytokinin ratio < 1 enhanced the growth rate of callus cultures; however, for 2,4-D the ratio was > 1.34–11.41 μM concentrations of growth regulators (IAA, NAA, BA and Kinetin) in the medium were found effective for callus induction and regeneration in all explants. The calli could be maintained over 32 months. BA (4.43 μM) combined with casein hydrolysate (10 mg l-1) yielded the highest rate of shoot production on hypocotyl (3–6) and cotyledon (5–7) explants and cotyledonary derived callus (4–8). More shoots were produced on explants cut from the most basal region of cotyledons from 5 to 7-day-old seedlings than from older seedlings or more distal cut sites. Apolar placement of explants, inhibited shoot regeneration. The shoot regeneration potential remained upto 7 months in calli developed on NAA + BA. Of three media tested, MS was superior to SH-M and B5. Rooting of shoots was not efficient; 42% of the shoots were rooted on MS medium containing sucrose (7–8%) + IAA (2.8–5.7 μM). Capitula induction was observed in both callus mediated shoots on cotyledons and shoots on rooting medium with sucrose, IAA, NAA and IBA. Well developed plantlets were transferred to the field with a 34% success rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of 1,2-benzisoxazole-3-acetic acid on adventitious shoot regeneration and in vitro rooting in apple

 The effect of 1,2-benzisoxazole-3-acetic acid (BOA), compared to 1-naphthaleneacetic acid (NAA), on adventitious shoot formation in leaf portions and compared to indolebutyric acid (IBA), on in vitro rooting in the apple (Malus domestica Borkh) cultivars McIntosh and Gala, and one rootstock, Jork 9, was investigated. BOA at 43.0 μm or 2.7 μm at NAA in combination with 17.8 μm benzyladenine (BA), induced the highest number of explants to produce adventitious shoots in Jork 9. In Gala, the combination of 21.5 μm BOA with 1.0 μm thidiazuron (TDZ) or with 22.0 μm BA induced the highest regeneration percentages, 58 and 54%, respectively, giving more satisfactory results than NAA (where only 42% of leaf explants exhibited shoot formation). In McIntosh, the highest percentage of regeneration was obtained with 1.3 μm NAA and 22.0 μm BA, while 51% was the highest response obtained with the BOA treatment. The combination of BOA with TDZ completely inhibited regeneration activity in leaf portions of this cultivar. The shoots of all the genotypes obtained with the most morphogenetic NAA or BOA treatments were excised, multiplied and successfully rooted and hardened. The results demonstrate that the synthetic auxin BOA is active in inducing shoot regeneration from leaf explants of apple and that the activity of BOA in plant regeneration is genotype dependent. When BOA was used to induce rooting in apple microcuttings, lower rooting percentages were obtained than with IBA, showing that the effect of BOA in inducing root formation is very low and that it cannot be used routinely to replace IBA in the in vitro rooting of microcuttings. Received: 18 June 1998 / Revision received: 4 January 1999 / Accepted: 29 January 1999     

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Parkia timoriana (DC.) Merr.: a multipurpose tree legume

In vitro regeneration of Parkia timoriana (DC.) Merr. has been achieved using cotyledonary node explants. The ability to produce multiple shoots has been evaluated using semi-solid Murashige and Skoog (MS) basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BA) either in single or in combinations. The explants cultured in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoots (96.66%) formation and number of shoots per explants (6.60), respectively. For rooting, full and half strength MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) and NAA were studied and the highest number of root formation was observed in full-strength MS supplemented with 9.8 μM IBA. Using Agrobacterium tumefaciens strain EHA105 pCAMBIA2301 various optimum conditions for efficient transformation were determined by recording the percentage of GUS⁺ explants. Following the optimized conditions, the co-cultured explants were cultured on semi-solid shoot regeneration medium containing MS medium + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime. After 8 weeks of culture, the regenerated shoots were rooted in rooting medium (RM) containing MS medium + 9.8 μM indole-3-butyric acid (IBA), 3% sucrose, 7.5 mg/l kanamycin and 500 mg/l cefotaxime. Successful transformation was confirmed by histochemical GUS activity of the regenerated shoots, nptII gene PCR analyses of the regenerated kanamycin resistant plantlets and Southern analysis of putative transgenic PCR⁺ plants.     

Micropropagation ofFagus sylvatica L.

Summary An in vitro shoot multiplication system was established from juvenileFagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.     

Plant growth regulators,adenine sulfate and carbohydrates regulate organogenesis and in vitro flowering of <Emphasis Type="Italic">Anethum graveolens</Emphasis>

In vitro regeneration protocol for Anethum graveolens (Apiaceae) was developed using leaf explants. MS basal medium used in experiments was augmented with various hormones for caulogenic and rhizogenic response. The optimum callus induction (100%) was obtained by leaf explants on MS media fortified with BA (0.5 mg l⁻¹) singly and in combination with NAA (0.1 and 0.2 mg l⁻¹). BA at 0.5 mg l⁻¹, KN at 1.0 mg l⁻¹ and NAA at 0.1 mg l⁻¹ induced highest number of multiple shoots (10.0 ± 0.25) per explant and they also showed in vitro flowering within 3 weeks of culture. Influence of adenine sulfate on regeneration frequency of callus was evaluated. The highest frequency of rooting (100%) with 6.0 ± 0.25 roots per explants was obtained in one-fourth strength MS medium supplemented with 1/4 MS + IBA 0.5 mg l⁻¹ within 4 weeks of transfer to the rooting medium. In vitro flowering (35%) was obtained with MS fortified with BA alone and also in combination with KN and NAA (5.3 ± 0.42 flowers per explants). In vitro flowering response was tested with different carbohydrates (fructose, glucose, mannose and sorbitol) and optimized. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. The proposed method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands of this medicinally important species.     

In vitro propagation of Petasites hybridus (Asteraceae) from leaf and petiole explants and from inflorescence buds

In vitro shoot regeneration from sterile leaf and petiole explants and from in-situ-collected inflorescence buds of Petasites hybridus was achieved by a simple two-step protocol. Murashige and Skoog (MS) nutrient medium was supplemented with 17.6 μm benzyladenine (BA)+0.54 μm naphthaleneacetic acid (NAA) to induce shoots. After 5 weeks of culture, 40% of the petiole and 27% of the leaf explants produced shoots compared to 76% of the inflorescence buds. Single shoots were excised and subcultured on MS medium supplemented with various cytokinins (N⁶-(Δ²-isopentenyl)adenine, BA, kinetin and thidiazuron). A concentration of 8.8 μm kinetin+0.54 μm NAA performed best in terms of shoot multiplication rate, average shoot length and spontaneous root induction. Received: 20 August 1997 / Revision received: 29 December 1997 / Accepted: 5 February 1998     

Soluble sugar,starch and phenolic status during rooting of easy- and difficult-to-root magnolia cultivars

The aim of the study was to determine the effect of indole-3-butyric acid (IBA) and exogenous sucrose concentrations on in vitro rooting, soluble sugar, starch and phenolic production, and ex vitro survival of four magnolia cultivars. There was a significant linear increase in rooting of most magnolia genotypes with an increase in IBA concentration in the medium from 1 to 6 mg L^?1. A further increase of IBA concentration to 10 mg L^?1 decreased (‘Elizabeth’, ‘Burgundy’) or had no effect on rooting frequency (‘Spectrum’). The effect of IBA on rooting of magnolia shoots was modified by sucrose supply. The three out of four magnolia cultivars showed the highest rooting efficiency in the presence of 6 mg L^?1 IBA and 30 g L^?1 of sucrose. Generally, decreasing and increasing the sucrose supply from 30 g L^?1 significantly lowered the rooting frequency. In ‘Yellow Bird’, sucrose at 40 g L^?1 totally blocked root formation. It has been found that the poor rooting ability of ‘Yellow Bird’ coincided with a low soluble sugar content, and high production of starch and phenolics in the shoot bases during the whole rooting period as compared to easy-to-root cultivars. After 5 weeks of the growth on IBA medium, rooted and unrooted shoots were transferred to ex vitro conditions. Both types of shoots showed a high survival and rooting rate (85.4–100%), but they differed in their growth activity and quality. Sucrose concentration in the rooting medium had a post-effect on ex vitro root formation and survival of magnolia plantlets. Ex vitro establishment (13.3%) of recalcitrant ‘Yellow Bird’ was obtained only when the shoots were taken from rooting medium containing the lowest level of sucrose (20 g L^?1).

     

Mother plant age and seasonal influence on in vitro propagation of <Emphasis Type="Italic">Quercus euboica</Emphasis> Pap., an endemic,rare and endangered oak species of Greece

A micropropagation method for Quercus euboica Pap. was developed. Nodal explants from seedlings gave higher multiplication rates than explants from adult plants. Cultures initiated at the beginning of May produced the highest percentage of shoot forming explants and multiplication rate. Woody Plant Medium (WPM) salts, with 100 mg l⁻¹ myoinositol, 1 mg l⁻¹ thiamine, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ nicotinic acid and 3% sucrose was used as basal medium and several cytokinins at various concentrations were evaluated for their effect on shoot multiplication. The highest shoot multiplication rate was obtained with 4.44 μΜ BA. IBA at 9.84 μΜ in the culture medium during the first week of culture, and if followed by culture in hormone-free medium, gave the best rooting results. Darkness at the beginning of the rooting period did not improve rooting. The use of plastic wrap as a cover material of the culture vessels enhanced rooting percentage and root number. Plantlets acclimatized ex vitro in soil from the natural environment of the species survived at a higher percentage (up to 93%) and had more vigorous growth than plantlets grown in a compost–perlite (2:1 v/v) medium (up to 36%).     

Effect of plant growth regulators,temperature and sucrose on shoot proliferation from the stem disc of Chinese jiaotou (<Emphasis Type="Italic">Allium chinense</Emphasis>) and in vitro bulblet formation

In vitro shoot proliferation from stem disc of Allium chinense, a vegetatively propagated plant, was investigated in this experiment. In the present study, shoots were formed directly on stem discs on a medium containing 1 mg/l N⁶-benzyladenine (BA) and 0.5 mg/lα-naphthaleneacetic acid (NAA). These shoots were further cultured on MS media supplemented with various levels of BA in combination with NAA, and new shoot clusters developed easily from the explants cultured despite considerable differences in the induction of shoot clusters with different levels of BA and NAA. The most productive combination of growth regulators proved to be 1.0 mg/l BA and 1.0 mg/l NAA, in which about 17 shoots were produced per cluster in 8 weeks culture. Most of the formed shoots were rooted 15 days after being cultured on MS media supplemented with 0.1–1.0 mg/l NAA. The survival rate of the plantlets under ex vitro conditions was 95% in pots filled with a peat: sand (2:1 v/v) mixture after two weeks. In vitro bulblet formation were strongly promoted by the high temperature of 30°C compared to that at 25, 20 and 15°C, and 12% (w/v) sucrose appeared to be optimal for bulblet development. Results from this study demonstrated that A. chinense could be in vitro propagated by using stem discs and in vitro bulblet formation could be achieved.     

In vitro multiplication and microrhizome induction in Curcuma aromatica Salisb.

Shoot multiplication and plant regeneration was achieved from freshly sprouted shoots of Curcuma aromatica on Murashige and Skoog's medium supplemented with BA alone (1–7 mgL^–1) or a combination of BA(1–5 mgL^–1) and Kn (0.5–1 mgL^–1). A concentration of 5 mgL^–1 BA was optimum for shoot multiplication and rooting of shoots. The regenerated plants grew profusely on transfer to liquid medium.In vitro raised plants were successfully established in the field. Microrhizomes were induced at the base of the in vitro derived shoots upon transfer to medium containingvarious combinations and concentrations of sucrose and BA and grown under varying photoperiods. MS basal medium with 5 mgL^–1 BA, 60 gL^–1 sucrose and an8 h photoperiod was optimum for induction ofmicrorhizomes within 30 days of culture. Harvestedmicrorhizomes stored in moist sand in poly-bagssprouted after 2 months of storage at roomtemperature. For in vitro storage, microrhizomeswere grown in medium containing 0.1 mgL^–1 BA.Microrhizome formation was found to be controlled bythe concentrations of BA and sucrose as well asphotoperiod during culture.     

Comparison of in vitro regeneration pathways in <Emphasis Type="Italic">Punica granatum</Emphasis> L.

When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM NAA, and 6 μM giberrellic acid (GA₃). However, adding 24 μM silver nitrate (AgNO₃) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l⁻¹ sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l⁻¹ sucrose.