The shoot regeneration frequencies changed with the variation of hormonal concentrations used in the same medium in this experiment . NAA at 1.0 mg/L in MS media showed the highest 80% ± 2.89% root initiation compared to IBA 70% ± 5.20% at the concentration of 1.0 mg/L . The increasing concentrations of IBA and NAA used in this experiment showed lower root initiation response . In the present study, an efficient regeneration method for native orange  was developed through in vitro culture technique. Very less work is done on this species specially in Bangladesh and that’s why a standard protocol for micropropagation is necessary to generate more disease-free plants of this species. Seed is an excellent source of explant to produce the fungus resistant plant because pathogen can’t reach to the seed level even after severe infection to the plant. Although regeneration in Citrus sp. is a very slow process, one of the major goals of the current study was to observe the effect of various plant growth regulators at different stages of regeneration from mature seed of Citrus reticulata. For callus induction of the native orange , 2,4-D was used as growth promoting hormone and seeds were used as explants in the current study. The result showed that 2,4-D was the most efficient plant growth regulator in callus induction. The absence of 2,4-D and presence of BAP, KIN in the culture medium initiated shoot induction from the callus as reported earlier in other species too. According to the report of , regeneration of shoot was found at various concentrations of BA from 0.5 – 4.0 mg/L for Citrus paradise  epicotyl explants. BAP 0.5 mg/l gave the best results  of shooting response in Citrus reticulata Blanco from different explants of in vitro raised seedlings according to the report of . The present study showed better regeneration response than those cited in these reports. NAA at 1.0 mg/L was determined as the major phytohormone for root initiation  in this experiment. A very similar result was observed in the earlier studies of. In the present investigation, we have established an efficient and simple protocol for the plant regeneration of C. reticulata using callus cultures induced from mature healthy seed explants.

This protocol may further be applied for genetic transformation in C. reticulata.Paclobutrazol [PBZ; common names: Bonzi, ClipperS, Clipper-T, Cultar, PP333; chemical name: IUPAC: -1-4–4,4-dimethyl-2-pentan-3-ol] is an efficient plant growth retardant listed for usage by the Plant Growth Regulation Society of America. Its optical enantiomer 2S,3S displays a pronounced plant growth regulatory activity,hydroponic fodder system whereas the 2R,3R enantiomer is more active in the inhibition of sterol biosynthesis, exhibiting fungicidal properties. The growth retarding form of PBZ  keeps certain structural similarities with entkaurene and ent-kaurenol, key compounds in the pathway to gibberellins  biosynthesis, and hence may inhibit cytochrome P450 monooxygenases, impairing the oxidation of ent-kaurene to ent-kaurenoic acid. As a result PBZ can cause a substantial depletion in the levels of active GAs throughout the plant, leading to a kind of dwarfism. Some effects of PBZ on plants are: development of dark green color in leaves; stimulation of flowering, likely as a result of a decrease in vegetative growth ; hastened leaf fall in autumn and emergence delay in spring in deciduous plants; yield increase in apples, Jathropa curcas and other cultivated species. Water solubility of PBZ is very poor, about 0.12 mM , the compound being mostly immobile in the phloem sieve tube elements. Due to that when sprayed in the plants its action is much localized. A more uniform distribution is seen when it is provided by trunk injection or soil application, because in this case the product is transported via xylem vessels. This low solubility has brought about some troubles especially when the compound is used under laboratory conditions. For instance in order to achieve reproducible results on the inhibition of seed germination of Townsville stylo  PBZ had to be autoclaved. The autoclaved product is also largely employed in aseptic culture experiments, although the non-autoclaved form is identically used . Sometimes the condition of the PBZ  employed in the experiments is not even mentioned. Since PBZ is “stable at all temperatures up to 50˚C for at least six months”and that during the autoclaving process temperature as high as 120˚C is achieved, it is likely that heating leads to degradation of the compound, rendering PBZ completely or partially inactive. As a consequence the effective amount of PBZ that elicits a physiological action is not known when the product is autoclaved. In order to throw some light to this problem a simple assay was designed as to compare the effects of autoclaved and non-autoclaved PBZ on the growth and dry mass accumulation of seedlings of sunflower . The first signs of PBZ action were observed by the development of a dark-green color in growing leaves 3 – 4 days after application of the product to the soil. The new appearing leaves then became smaller, exhibiting a thicker and wrinkled aspect. Thereafter the growth rate of PBZ-treated plants decreased substantially  and the apical internodes and leaves started displaying a rosette form. This dwarfism syndrome was similarly exhibited by autoclaved and non-autoclaved PBZ-treated plants, with no quantitative differences between the plants of the two treatments . Each one of those symptoms was properly reverted by GA3, the responses also not being affected by the autoclaving of PBZ. Stem growth pattern followed the well-characterized sigmoidal logistic model. As Figure 1 and Table 1 show stem length resulted much longer in the control and in all GA3-treated plants than in PBZ-inhibited plants. Again, differences in stem length in response to autoclaved and non-autoclaved PBZ were too small to have assumed any statistical significance. A dramatic change in the growth pattern of PBZ-treated plants was observed following GA3 application .

Growth rates increased substantially, sometimes higher than 10- fold ; in this way, at the end of the experiment the stem length of these plants surpassed that of the control non-treated plants. Leaf and internode number  were not affected by any of the treatments, showing that in sunflower they constitute highly conserved traits. Hence the much reduced stem length caused by PBZ , was a consequence of the very shorter internodes, an effect also reverted by GA3 notwithstanding whether PBZ was autoclaved or not . Total dry mass accumulated per plant did not respond to any of the treatment, autoclaved PBZ included . In this context, in some plant species GAs affect positively the net CO2 assimilation  whilst in others it is not affected.Sunflower thus, seems to belong to the latter group. This fact must be a consequence of a similar A displayed by PBZ-treated and non-treated sunflower seedlings. Similarly dry mass allocation to leaves and to the stem-root system was not affected either by PBZ or GA3 . The regulators, however, affected the dry mass partition within the stem-root system . Since leaf dry mass per plant was not affected by the regulators which also did not affect leaf number, the mean individual leaf dry mass was also kept similar amongst all the treatments . As a consequence of the similar total dry mass per plant and also of a similar leaf dry mass per plant, leaf mass ratio , the ratio between leaf to total plant dry mass, did not show any significant difference among the treatments . Though the mean individual leaf dry mass did not vary among the treatments, they profoundly affected leaf size as mentioned above. As described earlier leaf size was drastically reduced by PBZ, as also found in Zinnia and Geranium. In order to hold the same dry mass in a much reduced area PBZ-treated leaves have to be thicker, a point examined in a side experiment with the third uppermost leaves in the stem . Specific leaf area , was about 31.7 in control leaves, 26.5 – 28.8 in PBZ-treated leaves and 36.9 – 43.0 m–2·kg–1 in GA3-treated leaves. PBZ  thus induced the formation of small and thicker leaves, an effect promptly reverted by GA3. Shoot-root system dry mass as a whole did not respond to any of the regulators employed . Since total dry mass per plant was not affected as well, the ratio stem-root to total plant dry mass  was equally not affected by them . Within that system itself each one stem and root dry mass was, nevertheless, greatly affected . As shown in Figure 1, stem expansion was highly responsive to the regulators. Hence the system stem-root seemed to be the main target for the action of PBZ and GA3. When dry mass of stems and roots were considered separately they showed a large variation in response to treatments , PBZ  favoring dry mass allocation to roots and GA3 to stems. Hence the ratio root to total plant dry mass  was larger in PBZ-treated plant whereas stem to total plant dry mass  was larger in all  GA3-treated plants . In agreement with these observations, the root biomass accumulation was also increased in uniconazoland PBZ-treated plants of Arabidopsis.

Moreover these data are in close accordance with recent genetic studies which revealed good correlation between a lowGA regime and biomass accumulated in the roots. A pattern emerges as to explain the results on dry mass distribution in sunflower plants when the relationships between length and dry mass of the stem as affected by PBZ and GA3 are taken into account. It is well known that GA3 stimulates stem growth towards the shoot main axis direction which is translated into internode expansion . PBZ, on the other hand, inhibits stem elongation leading to much smaller cells. GA3-treated stems accumulate much more dry mass just because they are much longer than PBZ treated stems . By taking into account the dry mass distribution over the stem length unit, it is seen that both autoclaved and non-autoclaved PBZ-treated stems similarly “concentrate” more dry mass than GA3-treated stems , being thus “denser”. It is deduced that dry mass accumulation by individual cells of PBZ-treated plants may approach or even reach its potential, what did not absolutely occur in GA3-treated stems. Given that dry mass accumulation by the stem-root system does not vary with treatments  a model can then be proposed that fits to the above facts. In GA3-treated plants stem elongates excessively but dry mass accumulation  does not keep pace with that process, fodder system leading to the production of narrower and elongated stems. Nevertheless, due to this high elongation , stem constitutes the main sink of the stem-root system and thus in GA3- treated plants little dry mass remains to be allocated to the roots . A low root to shoot ratio  thus results . In PBZ-treated plants, stems are very short and although they accumulate more dry mass per volume unit , they are too small to constitute a strong assimilate sink. The surplus of assimilates produced by the plant is then directed towards the roots, what explains the high root to shoot  ratio displayed by PBZ-treated plants . Curiously, these calculations led to similar results as the ones obtained in with tomato using much more sophisticated techniques. By working with the A70  and W335  GA slow-growing mutants, those authors showed that the shoot constituted the main target for GA action. Accordingly, as growth in shoot elongation was much smaller in GA-deficient tomato plants much more assimilate was left to be partitioned into the roots as compared with the wild type plants. Summarizing, this dry mass accumulation pattern was due to stem growth as affected by GAs and not to be regulator by itself. Since total dry mass in sunflower plants did not respond to the regulators herein employed being kept more or less constant , a good theoretical exercise would be to forecast where the assimilates would come from in case the cells affected by GA3 would accumulated dry mass to their full potential. In summary when investigating the effects of autoclaving in PBZ physiological action on the growth and dry mass partition in sunflower seedlings, no significant difference in the inhibitory effects of autoclaved and non-autoclaved form was found and in the reversion of those effects by GA3, as well. Hence, the amounts  of autoclaved PBZ employed in aseptic cultures or used for other purposes in laboratory or field conditions could be considered as the real one and equivalent to the amounts of the non-autoclaved form.