In Vitro Propagation through Seed Culture and Regeneration of Asparagus Densiflorus L. through Callus Cultures Derived from Hypocotyls

Headnote

Abstract: A successful and reproducible clonal propagation protocol from seed culture and a high regeneration ability through callus cultures derived from hypocotyls of Asparagus densiflorus were achieved in this investigation. The results showed that good seed germination could be achieved by cold and wet seed stratification prior to culture and using sloped surface medium enriched with 2 mgl^sup -1^ BA. The use of hypocotyls was superior upon the use of radicals as explants for shoot multiplication. The best parameters for hypocotyl explants multiplication were recorded while using BA alone at 1.0 mgl^sup -1^ by producing 8.13 shoots/ explant of 7.81 cm mean length and 33.00 leaves per explant. The best auxin for rooting of Asparagus shoots was IBA at 1.25 mgl^sup -1^ as compared to IAA or NAA. At callus induction and initiation stage, a compact green callus was produced by the addition of 0.5+ 0.3 mgl^sup -1^ of both BA and NAA. The addition of 0.5+ 0.2 mgl^sup -1^ from BA and NAA gave the best callus regeneration ability. Using of BA and NAA at 0.5+ 0.2 gave the best shoots multiplication parameters regenerated from the callus. The highest number of roots (12.4 roots/ explant) and the longest roots (4.98 cm) were gained from the addition of 1.25 mgl^sup -1^ IBA. A high survival rate (95%) was found with Asparagus plantlets gradually acclimatized into the out-air conditions . Keywords: Asparagus densiflorus, micropropagation, in vitro seed culture, callus culture, regeneration

1. Introduction:

Asparagus plant Asparagus densiflorus belongs to Asparagus family Asparagaceae, is a native of South Africa is not really a fern at all. Even though it may appear fern-like to the inexperienced eye, it has flowers and berries, so is clearly an angiosperm. (Lemake, 2011). Asparagus densiflorus is a branching perennial herb with tough green aerial stems which are sparsely covered with spines. The leaves are actually leaf-like cladodes, which are 0.8-2 cm long and 0.1-0.2 cm wide, and arise in groups of four or more from the stem. Occurring in spring, the small white or pinkish-white flowers are 0.3-0.5 cm long and arise in clusters offthe stem. The root system is a mat of fibrous roots with bulbous tubers, from which plants may re-sprout (Wolf, 1999).

Asparagus densiflorus can be readily propagated by separating the tubers in fairly large clumps, or by sowing the seed in spring or early summer (Jamieson, 2002). Although Asparagus plant is regarded as an easy to propagate plant, but its propagation through the in vitro micropropagation technology is advantageous due to the production of high quality disease free, true-to-type plants independent of seasonal and other environmental conditions in a comparatively smaller space (Debergh and Zimmerman, 1991). Lateral bud and callus culture techniques are usually used for asparagus micropropagation (Matsubara and Clore, 1974). Benmousa et al. (1996) studied the optimization of callus culture and shoot multiplication of Asparagus densiflorus by testing different plant growth regulator concentrations. They found that the best treatment for callus initiation was on MS medium supplanted with 5.4 µM of pCPA combined with 4.4 µM of BA. A high number of shoots (3.3 shoots/ explant) was achieved from the use of 0.4 µM BA alone.

The present investigation aimed to test the in vitro propagation of Asparagus from stratified and nonstratifies seeds on plane or sloped MS media and testing various plant growth regulators at different levels. As well as testing the regeneration ability of the callus derived from hypocotyls.

2. Materials and Methods:

The current investigation was applied in Plant Tissue Culture laboratory of the Horticulture Department, School of Plant Production, Faculty of Agriculture and Forestry, University of Duhok, Iraq during the period from March, 2010 to November, 2010. Seeds of Asparagus were grouped into two groups, the first ones were stratified at low temperature (4° C) and high humidity and the second ones did not undergo any stratification treatments. The both groups of seeds were surface sterilized in mercuric chloride (HgCl2) at 0.1% for 15 minutes. They were then in vitro germinated on MS medium (Murashige and Skoog, 1962) supplemented with agar (7 g/l), sucrose (30 g/l), inositol (100 mgl-1), and thiamine-HCl (0.4 mgl-1). Furthermore, either 2 mgl-1 BA or 3 mgl-1 GA3 were added to the media which were prepared in two positions (slope or plane surface). Cotyl length, number of roots per seedling and mean length of roots were recorded after four weeks in culture. For shoots multiplication, Pieces of about 1 cm long of radilces and hypocotyls were inoculated on MS medium supplemented with different BA and NAA combinations (0, 0,3, 0.5 and 1.0 mgl-1 BA and 0.0, 0.1, 0.2 and 0.3 mgl-1 NAA). After six weeks from culture, the number of shoots and leaves per explant and the mean length of shoots were recorded. For rooting the produced shoots, different concentrations of IAA, IBA and NAA at 0.0, 1.0, 1.25 and 1.5 mgl-1 were tested and the number of roots per explant and mean length of roots were recorded after six weeks in culture.

In another experiment, callus was induced from the culture of hypocotyls on MS medium supplemented with 0.5 mgl-1 BA and 0.3 mgl-1 NAA. For callus regeneration, different concentrations of BA and NAA were tested (0.0, 0.3, 0.5 and 1.0 mgl-1 BA and 0.0, 0.1, 0.2 and 0.3 mgl-1 NAA). The same combinations were tested as well on the multiplication of shoots regenerated from the precious experiment. For rooting of the shoots, IAA, IBA and NAA were tested at different concentrations (0.0, 1.0, 1.25 and 1.5 mgl-1).

Ten replicates were assigned for each level of treatment and the experiment was designed according Completely Randomized Design (CRD). The comparison between means was carried out according to Duncan's multiple range test (P < 0.05) using a computerized program of SAS (SAS, 2001).

Finally, for acclimatization stage, a quite number of successfully rooted plantlets were removed from culture vessels and their roots were washed with distilled water and immersed in Benlate fungicide (0.1% for 10 min.). They were transferred to pots containing a steam sterilized soil mix (peatmoss+ loam+ Styrofoam 1:1:0.5, v:v:v) under tightly controlled atmosphere of the primary growth area.

3. Results and Discussion:

Asparagus in vitro seed germination was very successful in this investigation by producing healthy seedlings. Table (1) shows that the highest length of hypocotyls (6.32 cm), the highest number of roots per seedling (5.30) and the longest roots (4.68 cm) were recorded while culturing stratified seeds on a sloped surface MS medium supplemented with 2 mgl-1 BA. These results insure the importance of seed stratification. Stratification is the process of subjecting seeds to both cold and moist conditions. This process usually helps the seed to overcome dormancy caused by internal inhibitors like ABA by stimulating the release of gibberellins (Meyer, 2008). As well as the use of sloped surface media vs. the plane surface ones (Fig. 1, A and B) was found to be beneficial in plant tissue culture techniques by increasing the surface area of the medium which will enhance the performance of the cultured explant (George et al., 2008).

After seed germination, two kinds of explants (radicals and hypocotyls) were then used to show their ability to produce entire Asparagus plantlets by testing various BA and NAA combinations. Table (2) shows the results of using radical explants. The highest number of shoots per explant (5.20), the longest shoots (5.33 cm) and the highest number of leaves per explant (15.80 leaves/ explant) were recorded for the combined treatment 1.0+ 0.2 mgl-1 of both BA and NAA. This treatment did not show significant differences as compared to 1.0+ 0.3 mgl-1 treatment, but it was significantly higher than the rest treatments.

Table (3) shows the results of using Asparagus hypocotyl explants as affected by various BA and NAA combinations (Fig. 1, C). The best multiplication parameters were recorded while using BA alone at 1.0 mgl-1 by producing 8.13 shoots/ explant at a mean length of 7.81 cm and 33.00 leaves per explant. These findings are similar to those found by Debergh and Zimmerman (1991). These results confirmed that BA is the most important growth regulator for shoot multiplication, for which there is an absolute requirement. It is common to observe a relationship between BA concentrations and shoot number and shoot size (Chevreau et al., 1992). It can be noticed here that the use of hypocotyls as explants was better than the use of radicals.

According to the results of Table (3), the shoots produced from hypocotyl explants were chosen to undergo the rooting stage. At this stage, three kinds of auxins were tested (IAA, IBA and NAA) at different concentrations (Fig 1, D). Table (4) shows that IBA at 1.25 mgl-1 was the best auxin for Asparagus rooting (Fig 1, E) by producing 11.20 roots per explant at the length of 4.42 cm followed by NAA and IAA respectively. Such differences in the potency of auxin in inducing rooting might attributed to the structure of the auxins under study, the endogenous hormone level, as well as the genetic makeup of species under consideration (Toma, 2009).

A compact green colored callus was achieved from the culture of hypocotyls in MS medium supplemented with 0.5 mgl-1 BA and 0.3 mgl-1 NAA (Fig. 2, A). For callus regeneration, different concentrations of BA and NAA were tested. Table (5) shows that a good regeneration was achieved from these calli (Fig. 2, B). Since the highest number of shoots per explant (28.13) and the highest number of leaves per explant (34.50) were recorded for the combined treatment between BA and NAA 0.5+ 0.2 mgl-1 while the longest shoots (8.95 cm) were achieved for the combined treatment between BA and NAA 0.3+ 0.3 mgl-1. These results confirmed that the callus produced by Asparagus hypocotyls is an embryogenic callus. This confirms as well the necessity of auxins and cytokinins presence for callus cell differentiation and regeneration (Hartmann et al., 2002).

The same BA and NAA above combinations were retested at shoot multiplication stage. Table (6) shows that the best multiplication parameters were recorded while adding 0.5+ 0.2 mgl-1 from both BA and NAA respectively which gave 25.00 shoots per explant and 31.00 leaves per explant (Fig 2, C). Whereas the longest shoots (9.26 mgl-1) were achieved from 0.3+ 0.3 mgl-1 treatment.

For root formation stage of the shoots regenerated from the callus, the same three auxins (IAA, IBA and NAA) were tested. Table (7) shows that the best rooting parameters (12.4 roots per explant and the longest roots 4.98 cm) were recorded when 1.25 mgl-1 of IBA was added (Fig. 2, D).

At acclimatization and gradually moving into out-air conditions, a high survival rate was found with plantlets transferred to soil. Successful gradually acclimatized reached to 95%. This percentage was achieved by following hardening-offsteps carefully. Most of the plantlets began to grow well and did not show morphological abnormalities (Fig. 2, E).

References

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