EFFECT OF SOME BIOREGULATORS ON WHITE LUPINE (LUPINUS TERMIS) SEED YIELD AND ITS COMPONENTS AND ON ENDOGENOUS HORMONES CONTENT IN SEEDS

H.M. El-Saeid, Hussein Fawzy Abouziena, M.S.A. AbdAlla
Botany Department, National Research Center

ABSTRACT

A pot experiments was carried out at National Research Center, Dokki, Cairo, Egypt, to investigate the effect of IAA (100, 200 and 400 mg∙dcm^-3), NAA (10, 20 and 40 mg∙dcm^-3) and kinetin (25, 50 and 100 mg∙dcm^-3) on Lupinus termis plant growth, yield and seed chemical composition as well as hormones contents compared with untreated plants. The obtained results indicated that the highest number of branches was obtained with kinetin treatment, especially at 100 mg∙dcm^-3. While, the maximum values of both number and dry weight of pods were obtained as a result of foliar application with kinetin at 50 mg∙dcm^-3. Spraying the lupine plants with bioregulators increased significantly the seed number per pod and per plant, and seed yield/plant except those plants treated with IAA at 100 mg∙dcm^-3. Applications of IAA, NAA and kinetin treatments had no significant effect on crop index, harvest index and shelling percentage. The maximum seed yield was recorded with the application of kinetin at 50 mg∙dcm^-3 followed by kinetin at 100 mg∙dcm^-3 as well as IAA at 100 mg∙dcm^-3. High levels of GA and IAA were observed in seeds produced from plants treated by IAA relative to those treated by NAA or kinetin. Spraying the lupine plants with the bioregulators IAA, NAA and kinetin caused an increase in protein content and total carbohydrates and decrease of oil percent in the lupine seeds

Key words: lupine, IAA, NAA, kinetin, endogenous hormones, seed yield.

INTRODUCTION

White lupine (Lupinus termis Forsik) is one of the oldest agricultural crops widely used in the world not only as a protein source in fodder production but also for soil improvement [19]. Lupine belongs to the genus Lupinus in the Fabaceae family. Lupine seeds contain considerable nutrition due to its high protein (35-45%) and oil content (10-15%). In Egypt, the cultivated lupine area is about 1482 ha producing 2881 t with a productivity of 19,439 kg∙ha^-1 [10].

Plant growth regulators play important roles in the enhancement of the growth and productivity of the most economic crops. Gromadzinski et al. [13] reported that plant growth regulators resulted in significant increases in pod number, shortening of offshoots and seed yields of yellow and narrow-leaved lupines (Lupinus luteus and L. angustifolius, resp.) plants, but only when applied at the correct rate and date. Prusiński and Borowska [23] reported that sprayed plants of traditional yellow lupine cultivar with varied doses of the growth regulators auxin (indole-3-butritic acid) and cytokinin (6-benzyloaminopurine) limited the unfavourable abscission of generative organs, which resulted in a significantly higher seed yield, than the control.

Emery et al. [9] reviewed the literature and stated that a wide range of environmental factors can influence the extent of "flower shedding", and as a consequence the yield potential of many pulses may not be realized. Theories as to the cause include hypotheses based on competition for nutrients. They add that at different times during fruit development 18 different forms of cytokinin (CK) were detected in the component tissues [9]. El-Saeid et al. [8] reported that when cowpea (Vigna sinensis L.) plants were sprayed with IAA at the rate of 25 and 50 mg∙dcm^-3 increased number of leaves, shoot dry weight and number of produced flowers per plant and consequently significantly increased number and weight of pods and seeds per plant. Meanwhile 50 and 100 mg IAA significantly decreased the number of flowers abscised from cowpea plant. Seymour [26] reported that timing of application was critical, with application 6 weeks before flowering giving the most consistent positive responses. El-Bassiouny and Shukry [7] reported that foliar application of IAA at three concentrations (12.5, 25 and 50 mg∙dcm^-3) induced increments of the plant height, fresh and dry weights, number of branches and number of leaves per cowpea plant as well as yield components (pods per plant, seeds per pod, weight of seeds per pod and per plant and weight of seeds/fed) (fed = 0.42 ha). Khalil et al. [15] reported that kinetin treatments decreased stem height of lentil plant, however they induced significant increase in the number of leaves and branches, shoot dry weight and number of produced flowers per plant. Meanwhile, all treatment decreased the percentage of abscised flowers and increased number and weight of pods and seeds per plant.

The working hypothesis assumed that:

• spraying the bio-regulators have an effect on the productivity and chemical constituents features of lupine plants,
• there is a relationship between the endogenous hormones and lupine yield,
• the effect of bio-regulators on the plants varied depending on the concentration of the plant hormone application.

The aim of this investigation was to study the effect of IAA, NAA and kinetin applied at 3 concentrations on white lupine plants growth, yield, and chemical composition and hormones content in seeds.

MATERIAL AND METHODS

Two pot experiments were carried out in the greenhouse of the National Research Centre, Cairo, Egypt to study the effect of IAA (Indole-3-acetic acid) at 100, 200 and 400 mg∙dcm^-3; Naphthalene acetic acid (NAA) at 10, 20 and 40 mg∙dcm^-3 and kinetin at 25, 50 and 100 mg.dcm-3 and tap water (as control) on growth, yield and seed chemical constituent as well as hormones contents of white lupine (Lupinus termis Forsik) plants.

Seeds of white lupine cultivar Giza 1 were obtained from the Legume Research Section, Agriculture Research Center, Ministry of Agriculture, Egypt. Ten seeds were planted in plastic pots 50 cm diameter filled with 20 kg clay loam soil at 21^th of October in the two winter seasons and the plants were irrigated when needed. The seedlings were thinned at the age of 3 weeks to three uniform seedlings per pot. Triple superphosphate was mixed with the soil before sowing the seeds. The plants were fertilized with 3 g of nitrogen in the form of ammonium sulphate in three applications (one gram for each) after 4, 7 and 10 weeks from sowing.

Seedlings were sprayed with IAA at irrigated at 100, 200 and 400 mg∙dcm^-3; NAA at 10, 20 and 40 mg∙dcm^-3 and 25, 50 and 100 mg∙dcm^-3 in the case of kinetin and tap water as control. Tepole was added (1 mg∙dcm^-3 of growth substances) to the solution as wetting agent. The volume of spraying solution was maintained just to cover completely the plant foliage till drip. The plants were sprayed twice. The first spray was applied ten days before flowering as guided by the indicator plants and the second spray was performed ten days later. Other agricultural processes were performed according to normal practice.

Data recorded
At harvest, plant height, numbers of branches and pods per plant, pods weight, number of seeds per pod and plant, seed yield per plant, seed index (100 seed weight), shelling (%) [(seed weight per pod divided by pod weight) ×100], straw yield (g∙plant^-1), crop index (seed yield per plant divided by straw yield per plant), and harvest index (seed yield per plant divided by biological yield (above ground) per plant) were estimated. Furthermore, migration coefficient (pods dry weight divided by biological yield) was estimated according to the method described by Abdel Gawad et al. [2].

Chemical analysis
For determination of total carbohydrate, phenol sulphuric acid method was used according to Dubois et al. [6]. Nitrogen, element was determined according to the method described by Cottenie et al. [5]. Total protein was calculated using a conversion factor of 6.25. Oil content in the yielded seeds was determined as described by the methods mentioned in the A.O.C.S. [1] using petroleum ether (40-60°) in Soxhlet apparatus.

Endogenous hormone contents
Contents of endogenous IAA and GA₃ in lupine seeds were determined using High Performance Liquid Chromatography (HPLC). Extraction and purification were performed according to Jensen and Junttila [14] and Potts et al. [22] for IAA and GA, respectively. Analysis of samples by HPLC for IAA was carried out on a Perkin Elmer unit, Series (2), fitted with UV detector delivery system and analytical C18 reversed phase column. The solvent program was linear gradient (1%-99%) methanol in 1% acetic acid, the rate of the program (2% min^-1) at a flow rate 0.8 ml∙min^-1. Gibberellins were determined by the bioassay of lettuce hypocotyls in purified extracts chromatography [11].

Statistical analysis
A completely Randomized Design was used; each treatment was replicated six times. The obtained results were subjected to statistical analysis of variance according to the method described by Snedecor and Cochran [27] and the combined analysis of the two seasons was calculated according to the method of Steel and Torrie [28]. For comparison between means, L.S.D. test at 5% level was used.

RESULTS AND DISCUSSION

Plant height and number of branches
The data presented in Table 1 indicated that IAA increased significantly the white lupine plant height and the highest plant was recorded with 200 mg∙dcm^-3 of IAA. The auxin IAA increased linear growth of stem by causing cell elongation, due to greater osmotic uptake of water which results in increase of osmotic pressure or decrease in turger pressure or both. On the other hand, NAA and kinetin at 100 mg∙dcm^-3 treatments had a negative effect on the plant height.

Concerning the effect of the three growth regulators tested on number of branches per plant the results in Fig. 1 showed that IAA induced a lowest increment in most cases for the branches number, except the highest level. Regarding the effect of NAA the data in Fig. 1 showed no significant differences in branches number. The highest significant effect on branches number was obtained with kinetin treatment, especially the highest level. So, it could be conclude that kinetin treatments had more effect on branches number compared with auxins. Khalil et al. [15] found that kinetin treatments increased the dry weight of lentil shoots though they decreased stem length; such increase may be due to increase in number of branches and leaves. In this respect, Wickson and Thimann [30] reported that the application of CK reduced apical dominance and the lateral buds of intact plants which, otherwise, remain arrested could be made to grow by applying kinetin.

Figure 1. Effect of some bioregulators on number of branches and pods per white lupine plant

Yield and yield attributes criteria
Spraying the plants with growth substances increased both number and dry weight of white lupine pods per plant. It could be noticed that IAA at 200 mg∙dcm^-3 and kinetin at 50 and 100 mg∙dcm^-3 had a significant effect on pods number (Fig. 1), while pods dry weight per plant was significantly affected with all treatments, except NAA at 100 mg∙dcm^-3 (Table 1). The maximum values of both number and dry weight of pods were obtained as a result of foliar application with kinetin at 50 mg∙dcm^-3. In this respect, kinetin plays an important role in growth of the ovary by cell division [17]. Seymour[26] reported that lupine seed yield increases due to CK application were related to improvements in pod set.

Spraying the plants with bioregulators increased significantly the seed number per pod and per plant, and seed yield per plant, except those plants treated with IAA at 100 mg∙dcm^-3 (Table 1). The highest values of those criteria were obtained as a result of kinetin treatments, especially the medium level. Krishnamoorthy [17] reported that cytokinin influences the direction of transport of organic metabolites and minerals and their accumulation in cells. It's well known that metabolites are preferentially translocated to developing fruits and seeds. It's quite likely that phloem transport from source to sink may also be under the control of cytokinins. On the other hand, it could be noticed that seed index increased significantly as a result of growth substances treatments. Thus it can be concluded that the increment of seed yield per plant mainly due to the increase of seed number per plant. Prusinski et al. [24] found that weight of 1000 seeds and the seed yield of treated lupine plants with auxin were significantly higher than the non-treated control.

As for straw yield per plant, the increase was observed as a result of growth regulators treatments except kinetin at 50 and 100 mg∙dcm^-3 which decreased significantly the value of this character (Fig. 2). On the other side, these treatments had no significant effect on crop index, harvest index and shelling percentage. Generally, these treatments decreased those characters, except plants sprayed with kinetin. The highest values of crop and harvest index were obtained as a result of kinetin at 50 mg∙dcm^-3, while the maximum value of shelling percentage was obtained at 25 mg∙dcm^-3. The results showed also that migration coefficient was obtained under the effect of kinetin at 100 mg∙dcm^-3.

Figure 2. Effect of some bioregulators on seed and straw yields per white lupine plant

There was significant effect of plant growth regulators application on lupine seed yield. The maximum yield was recorded with the application of kinetin at 50 mg∙dcm^-3 followed by kinetin at 100 mg∙dcm^-3 as well as IAA at 100 mg∙dcm^-3 and minimum yield was recorded in unsprayed plants (Fig. 2).

The increment of seed yield as a result to application of CK could be attributed to that exogenous application of cytokinin to developing flowers prevents their abortion and permits the initiation of seed development [3]. Morris [21] reported that accumulation of cytokinins in developing seeds coincides with the highest rates of cell division, which is consistent with the idea that CK increase the sink strength of seeds for assimilates. Matthysee and Scott [20] stated that CK serve as a carrier from the root apex as it regulates the growth of lateral roots and lateral branches of the shoots. These results are in agreements with those reported by Seymour[26], Khalil et al. [15] and El-Saeid et al. [8]. Prusinski et al. [24] reported that the seed yield of yellow lupine treated with auxin was significantly higher than when treated with CK and the non-treated control.

Endogenous hormonal content
There is a relationship between the exogenous and endogenous hormones, especially GA3, and there is a relationship between the end product and endogenous hormones [4]. The results in Table (2) indicated that spraying the lupine plants with IAA or NAA at the three concentrations used caused a significant increase of the endogenous hormones contents i.e. GA and IAA in white lupine seeds. While application of kinetin at 50 and 100 mg∙dcm^-3 led to a significant increment of the two endogenous hormones (GA and IAA), however spraying kinetin at the low concentration (25 mg∙dcm^-3) had a slight enhancement of the endogenous hormones contents in lupine seeds.

It's evident that the level of IAA increased endogenous GA in the seeds, such increase were 362%, 218% and 166%, compared with control seeds when plants treated with 400, 200 and 100 mg∙dcm^-3 IAA, respectively. However the content of IAA in the seeds produced from plants treated by different levels of IAA was more than the control seeds. Such increases were 812%, 497% and 206%, when plants treated with 400, 200 and 100 mg∙dcm^-3 IAA respectively.

The amounts of GA in the seeds produced from plants treated by different levels of NAA are shown in Table 2. It's evident that the level of NAA increased endogenous GA in the seeds, such increase were 281%, 211% and 111%, compared with control. However the content of IAA in the seeds produced from plants treated by different levels of NAA was more than the control seeds. Such increases were 174%, 112% and 56%, when plants treated with 40, 20 and 10 mg∙dcm^-3 NAA, respectively.

Data in Table 2 indicated that sprayed white lupine plants with kinetin at concentrations of 25, 50 and 100 mg∙dcm^-3 resulted in significant increase the GA content in lupine seeds by 167%, 104% and 26%, relative to the unsprayed plants, respectively. In the same direction the amount of IAA in the seeds was increased by 147%, 91% and 26%, respectively in comparison to untreated plants. Similar finding was obtained by Khalil et al. [15], El-Saeid et al. [8]. Khalil et al. [15] found that Kinetin treatments increased the level of IAA, gibberellins and cytokine's in lentil plants.

It seems very fruitful to correlate and discuss changes in the separate endogenous groupings of active materials existing into extracted of seeds from plants treated by different levels of IAA, NAA and kinetin. The high levels of GA and IAA in seeds produced from plants treated by IAA relative to those treated by NAA or kinetin may explain the increase in plant height and number of leaves and pods. Its well known that the increase in the levels of both auxin and gibberellins promote cell division and cell enlargement and hence increased plant height [12]. On the other hand, high levels of auxins and gibberellins together were found to enhance leaf production [25].

Chemical contents in seeds
Data recorded in Table (2) revealed that spraying white lupine plants with the three growth substances i.e. IAA, NAA or kinetin at different concentrations caused an increase in resulted in increase of total carbohydrate as well as crude protein contents, compared to unsprayed plants. The highest significant values of total carbohydrates and protein contents were observed under the effect of kinetin at 100 and 50 mg∙dcm^-3 respectively. It's apparent from the data that fixed oil percent was decreased as a result of applied the three bioregulators treatments. So it could be concluded that there is a negative correlation between fixed oil and carbohydrate as well as protein content.

Using kinetin at 25, 50 or 100 mg∙dcm^-3 exhibited highly significant increments in the total carbohydrate and protein content and decreased the oil % rather than IAA, NAA and non-treated control treatments (Table 2). Similar findings were reported with Khalil and Mandurah [16], Tagade et al. [29] and Khalil et al. [15]. However Prusinski et al. [24] reported that auxin treatment had no significant effect on chemical composition or seed yield components of yellow lupine. Leopolda and Kriedeman [18] reported that kinetin plays an important role in protein synthesis through activation of nucleic acid synthesis, which needed phosphorus as participate part and potassium as stimulus to enzyme system.

CONCLUSIONS

1. Application of the three bioregulators IAA, NAA and kinetin increased the number of branches as well as pods and seeds per plant resulting in the significantly enhancement of seed yield per white lupine plant.
2. The maximum seed yield was recorded with the application of kinetin at 50 mg∙dcm^-3 followed by kinetin at the dose of 100 mg∙dcm^-3.
3. There was a negative correlation between fixed oil and carbohydrate as well as protein content.
4. Spraying white lupine plants with IAA or NAA at the three concentrations used and kinetin at 50 and 100 mg∙dcm^-3 caused a significant increase of the endogenous GA and IAA hormones contents in seeds.

REFERENCES

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