Journal of Experimental Botany, Vol. 53, No. 373, pp. 1437-1443,
June 2002
© 2002 Oxford University Press
Original Papers |
Inorganic nitrogen requirements during shoot organogenesis in tobacco leaf discs
School of Agriculture and Horticulture, University of Queensland-Gatton, Queensland 4345, Australia
Received 25 September 2001; Accepted 6 February 2002
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The role of nitrate, ammonium, and culture medium pH on shoot organogenesis in Nicotiana tabacum zz100 leaf discs was examined. The nitrogen composition of a basal liquid shoot induction medium (SIM) containing 39.4 mM
and 20.6 mM 
was altered whilst maintaining the overall ionic balance with Na+ and Cl- ions. Omission of total nitrogen and nitrate, but not ammonium, from SIM prevented the initiation and formation of shoots. When nitrate was used as the sole source of nitrogen, a high frequency of explants initiated and produced leafy shoots. However, the numbers of shoots produced were significantly fewer than the control SIM. Buffering nitrate-only media with the organic acid 2[N-morpholino]ethanesulphonic acid (MES) could not compensate for the omission of ammonium. Ammonium used as the sole source of nitrogen appeared to have a negative effect on explant growth and morphogenesis, with a significant lowering of media pH. Buffering ammonium-only media with MES stabilized pH and allowed a low frequency of explants to initiate shoot meristems. However, no further differentiation into leafy shoots was observed. The amount of available nitrogen appears to be less important than the ratio between nitrate and ammonium. Shoot formation was achieved with a wide range of ratios, but media containing 40 mM nitrate and 20 mM ammonium (70:30) produced the greatest number of shoots per explant. Results from this study indicate a synergistic effect between ammonium and nitrate on shoot organogenesis independent of culture medium pH. Key words: Ammonium, nitrate, organogenesis, tobacco.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Influences of the absolute and relative amounts of nitrate and ammonium on the induction and differentiation of plant cell cultures have been reported for a number of in vitro systems (Halperin and Wetherell, 1965
; Wetherell and Dougall, 1976; Chaleff, 1983; Grimes and Hodges, 1990; Cousson and Tran Thanh Van, 1993). In typical regeneration media the nitrogen pool is usually composed of two major components, inorganic nitrate and ammonium. Nitrate has been regarded as the principal form of nitrogen for the culture of plant tissue (Sathyanarayana and Blake, 1994) and in several studies could serve as the sole source of nitrogen for morphogenesis (Bayley et al., 1972; Wetherall and Dougall, 1976; Cousson and Tran Thanh Van, 1993). Drew et al. showed that localized nitrate, ammonium and inorganic phosphorus applications could stimulate root branching in barley (Hordeum vulgare) (Drew et al., 1973). Enhanced nitrogen metabolism was identified during de novo shoot organogenesis of tobacco callus (Hardy and Thorpe, 1990) and, in a follow-up study, enzyme and labelling data indicated that this occurred prior to meristemoid formation (Joy et al., 1994).
Ammonium used as the sole source of nitrogen appears to have a negative effect on growth and morphogenesis (Kirkby and Hughes, 1970; Cousson and Tran Thanh Van, 1993; Raab and Terry, 1994, 1995; Walch-Liu et al., 2000), although several studies have identified plants that can tolerate ammonium nutrition equally as well as nitrate (Oryza.sativa L.: Grimes and Hodges, 1990; Calluna vulgaris (L) Hull: Troelstra et al., 1995). The negative effect of ammonium has been attributed to various factors such as changes in medium pH and toxic effects of free ammonium. Tobacco cells are unable to utilize ammonium as a sole nitrogen source unless the medium is also supplemented with citrate, malate or pyruvate (Gamborg, 1970; Behrend and Mateles, 1975). By contrast, several studies have demonstrated that cell cultures can proliferate on media with ammonium as the sole nitrogen source if the pH of the medium is adjusted throughout the culture period and in the absence of an organic acid (Ipomoea and soybean: Martin et al., 1977; wild carrot: Dougall and Weyrauch, 1980).
In the experiments described in this study, the importance of balanced nitrogen nutrition for high frequency shoot organogenesis in tobacco leaf discs has been clarified. The influence of both nitrate and ammonium as sole nitrogen sources as well as in combination were evaluated with special emphasis placed on shoot meristem initiation. Media were also buffered with the organic buffer 2[N-morpholino]ethanesulphonic acid (MES) to examine nitrogen effects on organogenesis independent of pH.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Explant material and culture conditions
Seeds of Nicotiana tabacum L. var. zz100 were obtained from the Department of Primary Industries Southridge Research Station, Mareeba Queensland, Australia. Leaf discs, 7 mm in diameter, were aseptically removed from the leaves of in vitro-grown tobacco plants, using a cork borer and avoiding all major veins. Discs were floated with the ventral side in contact with a liquid shoot induction medium consisting of Murashige and Skoog nutrients (Murashige and Skoog, 1962
) supplemented with 3% (w/v) sucrose and 5 µM N6-benzyladenine. All cultures were of individual explants in 85x45 mm clear glass tissue culture jars (125 ml Pomade Jars, Cospak, Brisbane Australia) with white polypropylene screw capped lids and 25 ml of medium.
Culture jars were washed with great care using a two-step process. Firstly, jars were washed in a non-ionic detergent and rinsed in distilled water. Secondly, jars were soaked in 10% (v/v) perchloric acid for 5 d followed by thorough rinsing in ultra-pure water (R=18.2 M
cm-1; Milli-Q system, Millipore Australia Pty. Ltd., North Ryde NSW, Australia). Individual stock solutions of each macronutrient, micronutrient stocks, organic stocks, and hormone stocks were prepared using ultra-pure water in acid washed glassware and stored at 4 °C.
All treatments were replicated at least eight times and arranged randomly in a 25±2 °C incubator with 25–100 µmol m-2 s-1 of light from cool white fluorescent tubes (Polylux XL, F36W/840, General Electric, Great Britain) under a 12 h photoperiod. Experiments were independently replicated where indicated in the results section.
Maintaining an ionic balance in the culture medium
Prior to a survey of the effects of nitrate and ammonium on shoot organogenesis, an experiment was conducted to test the effect of using Na+ and Cl- as balancing ions when individual minerals were removed. For example, in this study the ion was supplied as NH4NO3 and KNO3. When was omitted from the culture medium, K+ and ions needed to be supplied in other forms in order to maintain an overall ionic balance. Using Cl- as a carrier ion, K+ was supplied as KCl and as NH4Cl.
After 30 d of culture, large increases in the concentration of Na+ and Cl- ions (from 0.2 mM up to 80 mM) in the culture medium had no significant effect on explant biomass, the proportion of leaf discs forming shoots or the number of shoots induced per disc compared to control levels (Ramage, 1999). Therefore, NaCl was used to supply Na+ and Cl- as accompanying ions in the manipulation of the nitrogen composition of culture media.
Buffering of culture media
The variation of nitrate- and ammonium-induced changes in medium pH during culture. In order to distinguish direct effects of mineral composition from indirect effects of medium pH, an organic buffer 2[N-morpholino]ethanesulphonic acid (MES) was used to establish a buffered shoot induction medium. Chemical buffers can affect the uptake of macronutrients by reducing the pH gradient through the plasma membrane. Therefore, various concentrations of MES were separately tested for effects on morphogenesis and growth of explants. Increasing the concentration of MES (up to 100 mM) in the shoot induction medium had no significant effect on explant biomass or the frequency of shoots induced (Ramage, 1999). MES at a concentration of 25 mM provided adequate buffering of culture media over 35 d (Ramage, 1999).
Variation of nitrate and ammonium supply
In the control shoot induction medium, nitrogen was supplied in two ionic forms, 39.4 mM and 20.6 mM , giving a total nitrogen concentration of 60 mM. The effects of and as sole sources of nitrogen were examined at six concentrations (0, 9.85, 19.7, 39.4, 49.7, and 60 mM ; and 0, 5.15, 10.3, 20.6, 30.9, and 60 mM ). In addition, ratios between the two forms of nitrogen were varied (:; 0:100, 30:70, 50:50, 70:30, 100:0).
Analyses
In all of the experiments, explants were cultured for 35 d, and the proportion of explants forming callus, meristems and/or leafy shoots recorded. In addition, culture media pH, the numbers of shoots per explant, and the fresh weights and dry weights of explants were recorded. When required, a log (base 10) transformation of fresh and dry weight data and a square root transformation 
Y+0.5 (where Y=the number of shoots) was performed to normalize data and to obtain homogeneity of the variances. Transformations were applied prior to analysis using PROC ANOVA or PROC GLM for analysis of variance in SAS (SAS Institute Inc.). The significance of differences between means was tested using the Duncan's Multiple Range Test and 95% confidence limits for transformed data were calculated using the T-method described previously (Sokal and Rohlf, 1981).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Selective removal of nitrate and ammonium
Nitrogen was supplied in two ionic forms,
and , in the control shoot induction medium (SIM) with nitrate constituting 66% and ammonium 34% of the total nitrogen pool (Table 1). The omission of total nitrogen or the selective removal of , whilst maintaining the ionic balance of the culture medium with Na+ and Cl-, inhibited the induction and formation of shoots, but the selective removal of did not (Table 2). Compared to SIM, the total nitrogen pool of media without ammonium was reduced from 60 mM to 39.4 mM. Although this constituted a 34% reduction in total nitrogen and resulted in a reduction in explant biomass and number of shoots per explant, nitrate as the sole source of nitrogen was sufficient for the induction and formation of shoots.
|
|
Nitrate as a sole nitrogen source
High frequency meristem formation was obtained after 35 d with as little as 9.85 mM nitrate (0.25 that in SIM) as the sole source of nitrogen in the culture medium, but the frequency of differentiation into leafy shoots was extremely low at around 12% (Table 3). Increasing the concentration of nitrate above 9.85 mM significantly increased explant biomass and enhanced the number of shoots per explant (P
0.01; dry weight Fs=79.11 and shoot number Fs=19.13, df=42, Table 3). Explants cultured on 39.4 and 49.7 mM nitrate produced the greatest numbers of shoots (53.88±12.23 and 50.38±7.10), but these were still significantly less than the control SIM medium (84.25±5.29; P0.01, Fs=19.21, df=84).
|
0.01).Interestingly, the pH of media at the end of culture was significantly affected by nitrate supply (P
0.01, Fs=13.86, df=42; Table 3
). The media from the 19.7, 39.4 and 49.7 mM treatments increased in pH from a mean of 5.75 (at the start of culture) to means after 35 d of 6.35, 6.26, and 6.20, respectively (
pH>0.4 units), but these were not significantly different from the control SIM medium (mean pH of 6.25±0.09 after 35 d culture; Fs=19.21, df=84, P
0.05).
The pH of media with 60 mM nitrate (5.88±0.15) did not vary from the pH at the start of culture and shoot numbers and explant biomass were significantly reduced (P0.01, shoot number Fs=19.13 and dry weight Fs=79.11, df=42), reflecting a deleterious effect of this concentration of nitrate (Table 3).
To examine the effect of nitrate supply on pH further, culture media containing 19.7, 39.4, and 49.7 mM of nitrate were chemically buffered to a pH of 5.8 prior to autoclaving with 25 mM of MES. Buffering media containing 19.7, 39.4, and 49.7 mM of nitrate significantly reduced the pH of culture media to a mean pH of between 5.4 and 5.6 after 35 d of culture (pH<0.3 units, P0.01, Fs=151.11, df=70; Fig. 1A). However, the addition of MES also significantly reduced explant biomass and the numbers of shoots per explant (P0.01, dry weight Fs=34.29 and shoot number Fs=11.51, df=35; Fig. 1B, C).
|
Ammonium as a sole nitrogen source
Ammonium as the sole source of nitrogen did not support the formation of meristems and shoots. Explants rapidly increased in diameter, changed colour from green to pale yellow or white, rarely produced callus (only 
5% of explants), and never formed adventitious organs. The most striking effect was a significant reduction in the pH of culture media over the 35 d culture period (P0.01, Fs=90.90, df=42; Fig. 2). Ammonium at concentrations 5.15 mM or greater resulted in pH levels up to 1.2 units below the initial pH, suggesting that the inhibition of growth and development maybe due to a pH effect.
|
-) and with ammonium as the sole source of nitrogen (-
-). Explants were cultured on liquid SIM media or with 20.6 mM ammonium as the sole nitrogen source. The ionic balance of the culture medium was maintained using Na+ ions. Data are the means from one independent experiment of n=8, ±1 standard error.To examine the effect of ammonium supply and pH on growth and development further, culture media with various concentrations of ammonium as the sole nitrogen source were chemically buffered to a pH of 5.8 prior to autoclaving with 25 mM MES. Buffering media containing 5.15, 10.3 and 20.6 mM of ammonium significantly reduced the pH of culture media, to a mean pH of between 5.4 and 5.5 at the end of culture (P
0.01, Fs=228.31, df=70; Table 4). Interestingly, 35–90% of explants cultured with ammonium as the sole source of nitrogen in a buffered media formed meristems that did not differentiate further into leafy shoots (Table 4). By contrast, explants cultured on a medium where ammonium constitutes 70% and nitrate 30% of the total nitrogen pool, formed shoots at an average of 57.38±8.10 shoots per explant and had a mean medium pH of 4.54±0.09 after 35 d (Table 5). Although shoot number was lower than the control shoot induction medium (57.38 versus 84.25 shoots per explant), this demonstrates that during shoot organogenesis there is a synergistic effect between ammonium and nitrate independent of culture medium pH.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In the shoot induction medium for N. tabacum L. leaf discs, the nitrogen pool was composed of two major components, inorganic nitrate and ammonium. Nitrate has been regarded as the principal form of nitrogen for the culture of plant tissues (Sathyanarayana and Blake, 1994
) and, in this study, could serve as the sole source of nitrogen for high frequency shoot formation. As little as 9.85 mM nitrate could induce high frequency meristem initiation in leaf discs. However, the proportion of explants that subsequently grew into leafy shoots was extremely low. Several other studies have also demonstrated the use of nitrate as the sole nitrogen source for morphogenesis (Bayley et al., 1972; Wetherall and Dougall, 1976; Cousson and Tran Thanh Van, 1993).
When nitrate was the sole source of nitrogen, culture media pH increased over the 35 d incubation period. A similar increase was observed in soybean suspension cultures with nitrate as the sole source of nitrogen (Bayley et al., 1972). Buffering the shoot induction medium with the organic acid MES limited the change in culture medium pH, but also resulted in a significant reduction in shoot number and growth (Fig. 1B, C). This result was in contrast to the three independent experiments that showed that 25 mM MES had no significant effect on shoot formation where both and were supplied; SIM media (Ramage, 1999). This suggests that, when nitrate is the sole source of nitrogen, nitrate supply is critical not pH per se. In this situation, the addition of MES stabilized the pH of culture media but may have affected the uptake/utilization of nitrate. However, when the source of nitrogen does not depend on nitrate uptake (i.e. both nitrate and ammonium are present) the control of pH may not be as critical. In this case, as the pH increased over the culture period, the alternative nitrogen source (ammonium) may have been utilized.
Ammonium used as the sole source of nitrogen in the shoot induction medium appeared to have a negative effect on explant growth and morphogenesis. No shoot meristem formation was observed in explants cultured with ammonium as the sole source of nitrogen. It has been reported that tobacco cells are unable to utilize ammonium as a sole nitrogen source unless the medium is also supplemented with citrate, malate or pyruvate (Gamborg, 1970; Behrend and Mateles, 1975). Furthermore, morphogenic studies of tobacco thin cell layers (TCL) by Cousson and Tran Thanh Van (1993) showed that when was the sole nitrogen source, the culture medium became acidified to a pH of approximately 4.2 during incubation. The authors suggest that this acidification of TCL culture media possibly increases K+ and Na+ influxes by the lowering of explant intracellular pH. An increase in the concentration of K+ from 20 mM to 80 mM resulted in the suppression of morphogenesis in 70% of the TCL explants. Although the morphogenic effect of Na+ and K+ described by these authors appears to corroborate this, changes in the internal concentrations of these ions were not determined or monitored.
Analysis of SIM media with ammonium as the sole nitrogen source revealed that the pH had dropped from the initial 5.75 to 4.5 by day 35 of culture. This raised the question of the role of culture medium pH during shoot formation. To examine this more closely, media containing ammonium as the sole source of nitrogen were buffered with 25 mM MES. Buffering prevented the drop in culture medium pH and shoot meristem initiation was observed in a number of explants. The frequency of initiation depended on the amount of ammonium added, but more importantly, meristems did not further differentiate into leafy shoots. This result supports the hypothesis that low pH, and not ammonium, was inhibiting the induction of shoot meristems. Furthermore, nitrate seems to be essential for the subsequent expansion of meristems into leafy shoots.
The role of organic acids, such as MES, in permitting the utilization of ammonium is unclear. Parfitt et al. suggest that MES functions primarily as a buffer against the acidification of the culture medium, as seen with ammonium as the sole nitrogen source (Parfitt et al., 1988). This is supported by several studies, which have demonstrated that cell cultures can proliferate on media with ammonium as the sole nitrogen source if the pH of the medium is adjusted throughout the culture period and in the absence of an organic acid (for example, wild carrot: Dougall and Weyrauch, 1980; Ipomoea and soybean: Martin et al., 1977). Results from this study are also consistent with this hypothesis, showing that shoot meristems can be initiated using ammonium as the sole nitrogen source when the medium is supplemented with MES.
Changes in medium pH as a result of nitrogen nutrition may be explained by a differential uptake of and 
The preferential uptake of at high pH causes medium acidification, which in turn results in the preferential uptake of and an increase in medium pH (Dougall, 1980; Congard et al., 1986; Martin and Rose, 1975). Perhaps as a consequence of this, the amount of available nitrogen appears to be less important for morphogenesis than the ratio between and 
Shoot formation was achieved with a wide range of ratios, however, a ratio of 70:30 (40 mM and 20 mM ) was optimal for shoot induction, with a significantly greater number of shoots induced per explant. Grimes and Hodges investigated the effect of varying the ratio of to on the regeneration of somatic embryos from rice (Grimes and Hodges, 1990). As found in this study, morphogenesis was achieved with a wide range of ratios and lowering the ratio from the control 80:20 (36 mM and 9 mM ) to 70:30 (31.5 mM and 13.5 mM ) resulted in a 1.5-fold increase in the mean number of plants induced per embryo (Grimes and Hodges, 1990). Similarly, Reinbothe et al. found that the optimal ratio of to was 11:1 in the production of somatic embryos from suspension cultures of Digitalis lanata (Reinbothe et al., 1990). Wetherell and Dougall also found that a reduced source of nitrogen (NH4Cl) was required to supplement nitrate levels for rapid growth and somatic embryogenesis of Daucus carota (Wetherell and Dougall, 1976).
To examine the role of pH in shoot induction further, it would be desirable to culture explants on a medium buffered to 4.5. MES is an effective buffer in the range pH 5.0–6.0 and is therefore not suitable (Parfitt et al., 1988). In addition, alternative buffers such as tris-(hydroxymethyl) amino have been shown to be phytotoxic to tobacco cultures (Parfitt et al., 1988). Furthermore, buffering a medium at this low concentration would affect the availability of many other minerals, which would complicate the interpretation of results.
The regeneration of adventitious shoots in vitro relies on the induction of shoot meristems and their subsequent growth into leafy shoots. It is not clear if the variation in shoot numbers observed in this study is a reflection of a variation in the numbers of shoot meristems induced, or whether changes in nitrogen supply affected the subsequent growth of meristems into leafy shoots without altering the numbers of meristems induced. Distinguishing between an increase in meristem number versus an increase in the growth and expansion of initiated meristems in this system is technically difficult. However, results from this study do demonstrate that omission or a change in the form and concentration of nitrogen during the early stages of shoot organogenesis inhibits the formation of meristems.
|
Acknowledgments |
|---|
We would like to thank the technical staff of the School of Agriculture and Horticulture. This work was supported in part by a UQ Collaborative Research Grant No. 97/QUALR3018G.
|
Notes |
|---|
1 To whom correspondence should be addressed. Fax: +61 75460 1283. E-mail: carl.ramage{at}mailbox.uq.edu.au
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bayley JM, King J, Gamborg OL. 1972. The ability of amino compounds and conditioned medium to alleviate the reduced nitrogen requirement of soybean cells grown in suspension cultures. Planta 105, 25.
Behrend J, Mateles RI. 1975. Nitrogen metabolism in plant cell suspension cultures. II. Role of organic acids during growth on ammonia. Plant Physiology 58, 510–512.
Chaleff RS. 1983. Induction, maintenance, and differentiation of rice callus cultures on ammonium as sole nitrogen source. Plant Cell, Tissue and Organ Culture 2, 29–38.
Congard B, Beaujard F, Viemont JD. 1986. Les bruyeres in vitro. VI. Croissance de Calluna vulgaris sur milieu strictement nitrique ammoniacal et cinetique du pH en fonction du development des plantes. Canadian Journal of Botany 64, 959–964.
Cousson A, Tran Thanh Van K. 1993. Influence of ionic composition of the culture medium on de novo flower formation in tobacco thin cell layers. Canadian Journal of Botany 71, 506–511.
Dougall DK. 1980. Nutrition and metabolism. In: Staba EJ, ed. Plant tissue culture as a source of biochemicals. Boca Raton, FL: CRC Press, 21–58.
Dougall DK, Weyrauch KW. 1980. Abilities of organic acids to support growth and anthocyanin accumulation by suspension cultures of wild carrot cells using ammonium as the sole nitrogen source. In Vitro 16, 969–975.
Drew MC, Saker LR, Ashley TW. 1973. Nutrient supply and the growth of the seminal root system of barley. I. The effect of nitrate concentration on the growth of axes and laterals. Journal of Experimental Botany 24, 1189–1202.
Gamborg OL. 1970. The effects of amino acids and ammonium on the growth of plant cells in suspension culture. Plant Physiology 45, 372–375.
Grimes HD, Hodges TK. 1990. The inorganic : ratio influences plant regeneration and auxin sensitivity in primary callus derived from immature embryos of indica rice (Oryza sativa L.). Journal of Plant Physiology 136, 362–367.
Halperin W, Wetherell DF. 1965. Ammonium requirement for embryogenesis in vitro. Nature 205, 519–520.
Hardy EL, Thorpe TA. 1990. Nitrate assimilation in shoot-forming tobacco callus cultures. In Vitro Cellular and Developmental Biology 26, 525–530.
Joy RW, Bender L, Thorpe TA. 1994. Nitrogen metabolism in cultured cotyledons of Pinus radiata during de novo organogenesis. Physiologia Plantarum 92, 681–688.
Kirkby EA, Hughes AD. 1970. Some aspects of ammonium and nitrate nutrition in plant metabolism In: Kirkby EA, ed. Nitrogen nutrition of the plant. Leeds: The Waverley Press, 67–76.
Martin SM, Rose D. 1975. Growth of plant cell (Ipomoea) suspension cultures at controlled pH levels. Canadian Journal of Botany 54, 1264–1271.
Martin SM, Rose D, Hui V. 1977. Growth of plant cell suspension cultures with ammonium as the sole source of nitrogen. Canadian Journal of Botany 55, 2838–2843.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.
Parfitt DE, Almehdi AA, Bloksberg LN. 1988. Use of organic buffers in plant tissue culture systems. Scientia Horticulturae 36, 157–163.
Raab TK, Terry N. 1994. Nitrogen source regulation of growth and photosynthesis in Beta vulgaris L. Plant Physiology 105, 1159–1166.[Abstract]
Raab TK, Terry N. 1995. Carbon, nitrogen and nutrient interactions in Beta vulgaris L. As influenced by nitrogen source, versus Plant Physiology 107, 575–584.[Abstract]
Ramage CM. 1999. The role of mineral nutrients in the regulation of plant development in vitro. PhD dissertation, The University of Queensland.
Reinbothe C, Diettrich B, Luckner M. 1990. Regeneration of plants from somatic embryos of Digitalis lanata. Journal of Plant Physiology 137, 224–228.
Sathyanarayana BN, Blake J. 1994. The effect of nitrogen sources and initial pH of the media with or without buffer on in vitro rooting of jackfruit (Artocarpus heterophyllus Lam). In: Lumsden PJ, Nicholas JR, Davies WJ, eds. Physiology, growth and development of plants in culture. The Netherlands: Kluwer Academic Publishers, 77–82.
Sokal RR, Rohlf FJ. 1981. Biometry. The principles and practice of statistics in biological research, 2nd edn. WH Freeman & Co.
Troelstra SR, Wagenaar R, Smant W. 1995. Nitrogen utilization by plant species from acid heathland soils. I. Comparison between nitrate and ammonium nutrition at constant low pH. Journal of Experimental Botany 46, 1103–1112.
Walch-Liu P, Neumann G, Bangerth F, Engels G. 2000. Rapid effects of nitrogen form on leaf morphogenesis. Journal of Experimental Botany 51, 227–237.
Wetherell DF, Dougall DK. 1976. Sources of nitrogen supporting growth and embryogenesis in cultured wild carrot tissue. Physiologia Plantarum 37, 97–103.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||