Docstoc

Chapter 4

Document Sample
Chapter 4 Powered By Docstoc
					4

Establishing Micropropagation Protocols for Four Watsonia Species

An unanswered question is a fine traveling companion. It sharpens your eye for the road.
Rachel Naomi Remen

4.1

INTRODUCTION

Micropropagation is an important tool that facilitates research and development in many fields of plant science. Not least of these is propagation of ornamental plants, where tissue culture has allowed mass propagation of superior genotypes and allowed fundamental research to be conducted on the physiology of morphogenesis, growth and reproduction of these plants. Here we review and study factors that impact the micropropagation of several Watsonia species that have the potential to be new ornamental crops.

4.1.1 Explant selection

In vitro culture of Gladiolus, a close relative of Watsonia, was first reported by ZIV, HALEVY and SHILO (1970) who used inflorescence stalks as explants. Since then, various other plant parts have been used as explants, including flowers, bracts, perianth, seedlings, corms and leaves. Work on appropriate explant selection for in vitro culture of Watsonia has not been conducted.

4.1.2 Proliferation in tissue culture

Auxins are commonly used to initiate shoots on explants (HUSSEY, 1977b) and induce callus formation in many species. Naphthaleneacetic acid (NAA) produces a non-friable callus (KAMO, CHEN and LAWSON, 1990), while 2,4-

dichlorophenoxyacetic acid (2,4-D) produces callus that can be used for suspension culture (REMOTTI and LÖFFLER, 1995). High concentrations of cytokinins are useful for shoot proliferation (ZIV, 1989) although this may lead to thicker, shorter and distorted shoots having no roots (HUSSEY, 1976c). Cytokinins are also used in protoplast culture (TAYLOR, BHATTI, LONG and SAUVE, 2000) and embryogenic
89

Micropropagation

suspensions (REMOTTI, 1995). Cytokinins are reported to inhibit cormlet formation in liquid cultures (STEINITZ and LILIEN-KIPNIS, 1989). Multiplication is usually by precocious proliferation of existing axillary shoots that occur in the axil of every leaf (HUSSEY, 1976c).

Growth retardants such as ancymidol (ZIV, 1989), paclobutrazol (PP3) (LIPSKY, KATAEVA and BUTENKO, 1997), daminozide and uniconazole (ZIV, 1990) have been used as inhibitors of gibberellin synthesis to reduce shoot growth and induce meristemoids (bud clusters) in liquid cultures (STEINITZ and LILIEN-KIPNIS, 1989). Buds cultured without growth retardants develop leaves that continue to elongate in the liquid medium, but rapidly become hyperhydric (ZIV, 1989; 1990; 1991a). Liquid culture systems used for micropropagation have lead to increased growth and multiplication rate of shoots, roots, storage organs and somatic embryos (ASCOUGH and FENNELL, 2004). In Gladiolus, liquid-shake cultures have improved growth rates of shoots (NHUT, TEIXEIRA DA SILVA, HUYEN and PAEK, 2004), meristematic clusters (ZIV, 1991a), and corms (DANTU and BHOJWANI, 1995).

4.1.3 Plantlet survival ex vitro

Successfully transferring in vitro generated plantlets to the ex vitro environment is a critical step in the micropropagation process, as large losses can be incurred if plant survival rate is low. An in vitro rooting stage is often performed prior to plantlets being planted out. In tomato hypocotyls, white light is required for root initiation in vitro (TYBURSKI and TRETYN, 2004), while for Eucalyptus, darkness has been used as a pretreatment to initiate rhizogenesis (BALTIERRA, MONTENEGRO and DE GARCIA, 2004). The effect of auxins on root initiation is well known, and other factors known to improve in vitro rooting include galactomannans (LUCYSZYN, QUORIN, RIBAS and SIERAKOWSKI, 2006), polyamines (ARENA, PASTUR, BENAVIDES and CURVETTO, 2005), activated charcoal (PAN and VAN STADEN, 2002), gibberellic acid (LE GUEN-LE SAOS and HOURMANT, 2001) and cyclodextrins (APOSTOLO, BRUTTI, FERRAROTTI, LLORENTE and KRYMKIEWICZ, 2001). Compounds that inhibit rooting include riboflavin (ANTONOPOULOU, DIMASSI, THERIOS, CHATZISSAVVIDIS and TSIRAOGLOU, 2005), ammonium nitrate (BENNETT, MCDAVID and MCCOMB, 2003), and aluminum (BOJARCZUK, 2000). For some species, such as the carob tree, high sugar concentrations improve rooting
90

Micropropagation

(CUSTODIO, MARTINS-LOUCAO and ROMANO, 2004), while in others like avocado, it has a negative effect (PREMKUMAR, BARCELO-MUNOZ, QUESADA, MERCADO and PLIEGO-ALFARO, 2003).

Here we study the factors affecting culture initiation, shoot induction and multiplication, growth and morphogenesis in liquid culture, in vitro rooting, and ex vitro acclimatisation.

4.2

MATERIALS AND METHODS

4.2.1 Culture initiation

Seeds of four Watsonia species (W. gladioloides and W. lepida from the summer rainfall area and W. laccata and W. vanderspuyiae from the winter rainfall area) were obtained from Silverhill Nurseries, Kenilworth, South Africa. Seeds were surface sterilised for 15 min in a 50% (v/v) commercial bleach (Jik®) solution (3.5% NaOCl) with Tween 20 as a surfactant. Seeds were rinsed three times with sterile distilled water, placed on a 1/10 strength MS (MURASHIGE and SKOOG, 1962) medium with 100 mg l-1 myo-inositol, 0.9% agar (Agar-Agar No. 1, Marine Chemicals, India), but without hormones or sucrose, sterilized by autoclaving at 121 ° and 103.4 kPa for C 20 min. For germination, seeds of W. gladioloides and W. lepida were incubated at 25 ° while seeds of W. laccata and W. vanderspuyiae were incubated at 15 °C, C, based on their optimum germination temperatures previously observed (Chapter 3). For subsequent experiments, individual shoots were grown in 33 ml cylindrical culture tubes containing 10 ml of medium. All tubes were capped and then sealed with Parafilm® and incubated at 25 ± 1 °C under Osram ® 75 W cool white fluorescent tubes providing a 16-h photoperiod and a light intensity of 12.6 µmol m-2 s-1 at culture level.

4.2.2 Effect of hormones and explant type on adventitious shoot induction

Seedlings that germinated successfully were selected for uniformity based on height (8 cm) and leaf number as some seedlings produced multiple leaves immediately after germination. At this time, selected seedlings were removed and divided into three sections: the root, hypocotyl and leaf. These were placed on MS media
91

Micropropagation

containing 3% sucrose, 100 mg l-1 myo-inositol, 0.9% agar with one of the following hormone combinations: control (no hormones), 0.5 mg l-1 BA, 1 mg l-1 BA, 0.5 mg l-1 NAA, 1 mg l-1 NAA, 0.5 mg l-1 BA + 0.5 mg l-1 NAA, 1 mg l-1 BA + 0.5 mg l-1 NAA, 0.5 mg l-1 BA + 1 mg l-1 NAA, 1 mg l-1 BA + 1 mg l-1 NAA. Ten replicates were done for each explant type of each species for each treatment. After 60 d, the number of shoots produced by each explant in each treatment was recorded.

4.2.3 Effect of BA and NAA on shoot multiplication on solid media

An individual clone was selected from each species at the conclusion of the above study, and used for bulking up (MS medium with 0.5 mg l-1 BA) so that genetic differences could be minimised. The effect of BA and NAA on shoot multiplication were investigated by subculturing individual intact shoots onto MS media containing 3% sucrose, 100 mg l-1 myo-inositol, 0.9% agar with one of the following hormone combinations: control (no hormones), 0.5 mg l-1 BA, 0.5 mg l-1 BA + 0.5 mg l-1 NAA, 1 mg l-1 BA + 0.5 mg l-1 NAA or 0.5 mg l-1 BA + 1 mg l-1 NAA. Five replicates were done for each treatment and the experiment was repeated twice. The mean number of shoots per explant was recorded after 30 d.

4.2.4 Effect of liquid and solid media on multiplication and growth index

To determine the effect of a liquid growth medium on shoot multiplication and growth index (proportionate gain in weight), intact shoot explants were subcultured onto either solid (solidified using 0.9% agar) or liquid media (MS with 3% sucrose and 100 mg l-1 myo-inositol). Erlenmeyer flasks (100 ml) were used for the liquid cultures and rotated at 120 rpm on a Janke & Kunkel KS 500 rotary shaker. Five replicates were done for each treatment and the experiment was repeated twice. The treatments consisted of a control (no hormones) and 0.5 mg l-1 BA. After 30 d, the number of shoots produced by each explant was recorded. Shoot fresh weight was determined at the beginning and end of the experiment, and the growth index (GI) was calculated as follows: (final weight – initial weight) / initial weight (ZIV, 1990).

92

Micropropagation

4.2.5 Effect of media strength and hormones on meristemoid induction

Intact shoots were subcultured onto either solid (solidified using 0.9% agar) or liquid media (MS or half-strength MS with 3% sucrose and 100 mg l-1 myo-inositol). Erlenmeyer flasks (100 ml) were used for the liquid cultures and rotated at 120 rpm. Five flasks were done for each treatment, each flask contained three shoot explants. The experiment was repeated twice. Treatments consisted of a control (no hormones), 5 mg l-1 BA and 5 mg l-1 PP3. Growth index was calculated for all explants and the number of explants that produced meristemoids was noted.

4.2.6 Effect of auxin type and concentration on in vitro rooting and ex vitro acclimatisation

To determine the effect of exogenously applied auxins on root formation and subsequent ex vitro acclimatization, intact individual shoots were subcultured onto MS media (3% sucrose, 100 mg l-1 myo-inositol and 0.9% agar) containing either 0.1 mg l-1 or 1.0 mg l-1 of the following auxins: IAA, IBA, NAA, PAA and 2,4-D. Five explants were used for each treatment, and the experiment was repeated three times. After 30 d, the number of roots was recorded for each explant and data analysed. Plantlets were placed in seedling trays in vermiculite and moved to the misthouse where acclimatization took place. After an additional 30 d, the number of surviving plantlets was recorded.

4.2.7 Data analysis

Explants were randomly assigned to treatments, and data were recorded as means of at least five replicates, with each experiment being repeated at least twice. Since these repeated experiments were initiated at the same time, they were considered as separate blocks, and the means from each treatment were used for analysis. Data were analysed for significant differences using analysis of variance (ANOVA) and means were separated using Fisher’s individual error rate at the 5% level of significance using Minitab® Release 14.

93

Micropropagation

4.3

RESULTS AND DISCUSSION

4.3.1 Culture initiation

Seeds began germinating within seven days of being placed on the germination medium. Although germination was not simultaneous, maximum germination occurred within three weeks. Those seeds that did not germinate within three weeks did not germinate even after an additional three weeks, and were therefore considered nonviable. Very little fungal contamination was observed, suggesting that decontamination was mostly successful.

4.3.2 Effect of hormones and explant type on adventitious shoot induction

Shoot induction from seedling explants was successful only when the hypocotyl region of the seedling was used (Table 4.1). Although root explants produced more roots, only 5% from W. laccata produced shoots. Leaf explants were completely unresponsive, except for a small percentage of those from W. vanderspuyiae. The data presented are the combined results from all treatments, and no significant differences between treatments were found.
Table 4.1:Shoot induction from Watsonia seedling explants (%). Values represent pooled results from all treatments since responses were identical. Means with different letters within a row are significantly different (P < 0.05). Species W. gladioloides W. lepida W. laccata W. vanderspuyiae Root 0b 0b 5b 0b Hypocotyl 80 a 94 a 80 a 98 a Leaf 0b 0b 0b 7b

In all treatments, and across all species, hypocotyl explants were primarily capable of regenerating shoots. Except for W. vanderspuyiae, leaf explants showed no response at all, not even callus initiation, while root explants continued to produce roots. This is probably because in most monocotyledonous species the meristem is located at the base of the seedling, and would continue producing the shoot when excised. As mentioned before, explant source determines the relative success of in vitro culture (NHUT, TEIXEIRA DA SILVA, HUYEN and PAEK, 2004). ZIV and
94

Micropropagation

LILIEN-KIPNIS (2000) showed that greater numbers of shoots were formed on Gladiolus inflorescence explants compared to corm explants. Although there may be some question about the use of non-genetically-uniform seedlings, the consistent response both across and within treatments suggests that these data result from differences in the anatomy of the explant (presence of the apical meristem) as opposed to genetic or physiological differences. However, the lack of a response from such young seedling tissue suggests there may be mechanisms restricting totipotency, or perhaps differentiation in these plants is irreversible. Consequently, studies on cell division and regeneration of subsections of the hypocotyl were performed in an attempt to understand this phenomenon (see Chapter 6).

As with Gladiolus (DICKENS, KELLY, MANNING and VAN STADEN, 1986), Freesia (HUSSEY, 1977b) and Dierama (MADUBANYA, 2004), maximum shoot induction for Watsonia required both an auxin and a cytokinin present in the medium (Figure 4.1 and Figure 4.2).

4.3.3 Effect of BA and NAA on shoot multiplication on solid media

The highest shoot multiplication for all species occurred when explants were cultured on media containing 0.5 mg l-1 BA (Figure 4.3). For W. gladioloides, a BA:NAA ratio of 0.5:1 also significantly increased shoot multiplication. For W. lepida, all hormone combinations significantly increased shoot multiplication compared to the control.

For W. laccata, a BA:NAA ratio of 0.5:0.5 improved shoot multiplication. For W. vanderspuyiae, shoot multiplication was increased in all treatments except when a BA:NAA ratio of 1:0.5 was used. For Gladiolus, BA is usually used for successful shoot multiplication (HUSSEY, 1976c; ZIV and LILIEN-KIPNIS, 1997). Interestingly, in Dierama latifolium, maximum shoot production occurred when only NAA was present in the medium (PAGE and VAN STADEN, 1985) while for Dierama luteoalbidum, BA alone resulted in maximum shoot multiplication (MADUBANYA, 2004). This does not appear to be the case for Watsonia, as for all species tested, maximum shoot proliferation occurred when BA was the only hormone in the medium.

95

Micropropagation

96

97

Micropropagation

8 a Control 0.5 mg/l BA BA:NAA = 0.5:0.5 BA:NAA = 1:0.5 BA:NAA = 0.5:1

Mean number of shoots per explant

6

a b b a a b b c c c b b b b a a a

4 b

c 2

0

g W.

la

es loid dio

lep W.

ida

lac W.

ca

ta va nd

pu ers

yia

e

W.

Figure 4.3: Effect of NAA and BA on shoot multiplication of four Watsonia species using agarsolidified MS medium. Bars with different letters indicate significant differences between treatments within a species (P < 0.05).

4.3.4 Effect of liquid and solid media on multiplication and growth index

The effect of liquid media and the presence or absence of BA on shoot multiplication was inconsistent among the four species (Figure 4.4 and Figure 4.5). On a solid medium, BA significantly increased shoot multiplication in all species, but in a liquid medum, this was only true for W. lepida and W. laccata. Without BA, shoot multiplication was significantly increased in a liquid medium in all species except W. lepida. The effect of media type and growth hormones on GI are similar across Watsonia species (Figure 4.6).

For W. gladioloides and W. vanderspuyiae, presence or absence of BA was not a significant factor, but the type of medium was – the GI was higher in liquid cultures. For W. lepida, the trend was similar, but in a liquid medium, BA acted synergistically and resulted in an increased GI. For W. laccata, GI was significantly increased when BA was present in the medium (liquid and solid).

98

Micropropagation

Watsonia species showed different responses to medium type (liquid or solid). For W. gladioloides and W. vanderspuyiae, explants on a solid medium produced significantly more shoots than those in liquid medium, while no difference was found between solid- and liquid-grown explants of control W. lepida (Figure 4.4). This situation is different from the many other species where liquid culture greatly improves propagule production (ASCOUGH and FENNELL, 2004).

10

a

Mean number of shoots per explant

8

Solid control Liquid control Solid + 0.5 mg/l BA Liquid + 0.5 mg/l BA

b

6

a a

4

b c

c b

ab a c b c

2

c

c

c

0

g W.

i iolo lad

s de

a pid . le W

c lac W.

ata v W. d an

iae uy sp er

Figure 4.4: Shoot multiplication of Watsonia species on agar-solidified and liquid MS medium with or without 0.5 mg l-1 BA. Bars with different letters indicate significant differences between treatments within a species (P < 0.05).

In all four species examined here, maximum growth occurred in a liquid medium (Figure 4.6). This is well documented and probably resulted from a combination of factors including greater availability of water (DEBERGH, 1983), better nutrient availability, and a reduction in nutrient and hormone gradients (GAWEL and ROBACKER, 1990). These beneficial effects are due to the lower resistance to diffusion and closer contact between explant and culture medium (SINGHA, 1982; cited by AVILA, PEREYRE and ARGÜELLO, 1996). In some cases, improved growth rate in liquid culture is due to greater water uptake, but in others, carbohydrate and organic nitrogen accumulation is enhanced, suggesting that nutrient assimilation is favoured in liquid culture (AVILA, PEREYRE and ARGÜELLO, 1996).
99

Micropropagation

100

Micropropagation

50
a
Solid Control Liquid Control Solid + 0.5 mg/l BA Liquid + 0.5 mg/l BA

40 Mean Growth Index
a

30
b a

20

ab bc

10
c c

c b bc c

a

a

b

b

0
W. g io lo la d s ide le W. a p id la W. cca ta van yi spu der ae

W.

Figure 4.6: Effect of solid and liquid media with or without 0.5 mg l-1 BA on the mean growth index of Watsonia species. Bars with different letters indicate significant differences between treatments within a species (P < 0.05).

In all treatments of W. laccata and W. lepida incubated in a liquid medium containing 0.5 mg l-1 BA, the GI increased concomitant with increased shoot multiplication. For the other species, this was not the case, and increased shoot multiplication did not translate into an increased GI.

4.3.5 Effect of media strength and hormones on meristemoid induction

Meristemoid (bud cluster) formation was inconsistent among the four Watsonia species (Table 4.2, Figure 4.7). Without growth regulators, very few or no meristemoids formed on shoot explants. A half-strength MS medium containing 5 mg l-1 BA resulted in meristemoid formation in all species, albeit at a low frequency. Addition of 5 mg l-1 PP3 in a half-strength MS medium induced meristemoid formation in all species except W. vanderspuyiae.

In the two species from the summer-rainfall region, W. gladioloides and W. lepida, a full-strength MS medium significantly increased growth of explants compared to those on a half-strength medium. In contrast, species from the winter rainfall area, showed no difference in explant growth in half-strength or full-strength MS media.
101

Micropropagation

102

Micropropagation Table 4.2: Effect of media strength and growth regulators on meristemoid formation (%) on Watsonia shoot explants. Species W. gladioloides W. lepida W. laccata W. vanderspuyiae Note: A: Treatment 1 - ½ MS control; 2 – MS control; 3 - ½ MS + 5 mgl BA; 4 – MS + 5 mgl BA; 5 - ½ MS + 5 mgl PP3; 6 - MS + 5 mgl PP3.
-1 -1 -1 -1

Treatment 1 10 0 0 0 2 0 0 0 10 3 10 20 10 50

a

4 11 33 56 0

5 50 30 33 0

6 40 0 0 56

Although meristemoids were produced in all four Watsonia species tested in this study, their formation was inconsistent and unpredictable with respect to type of treatment. In some instances, high numbers of meristemoids were formed from explants that exhibited reduced GI in response to PP3 or reduced medium strength, while in contrast, high concentrations of BA induced meristemoid formation without reducing GI. No significant trend was found between mean GI and meristemoid formation when regression analyses were conducted (Table 4.3), but these negative findings do not necessarily imply that no trend exists.
Table 4.3: r2 and F-probability values for regression of mean growth index on percent meristemoid formation of Watsonia liquid cultures. Species W. gladioloides W. lepida W. laccata. W. vanderspuyiae R2 0.37 0.76 0.54 0.24 F-probability 0.5 0.12 0.31 0.66

In Nephrolepis liquid cultures, inhibition of leaf growth led to meristemoid induction, while in Gladiolus PP3 was required to reduce shoot growth and so allow meristemoids to form (ZIV, 1991a). This suggests that suppression of leaf growth is necessary to induce meristemoids, which may result from a redirection of assimilates used in shoot elongation to formation of additional apical domes.

103

Micropropagation

4.3.6 Effect of auxin type and concentration on in vitro rooting and ex vitro acclimatisation

The effect of applied auxins on the stimulation of rooting in vitro was pronounced, and trends were similar across the four species examined here (Figure 4.8). In all four species, IBA and NAA at a concentration of 0.1 mg l-1 significantly increased mean number of roots per explant. Interestingly, in all four species except W. laccata, addition of IAA and NAA at a concentration of 1 mg l-1 increased root number, but IBA at this level produced the same number of roots as the control.

10

30

Mean number of roots per explant

a A
8

b B a a A A* B
25 20 15

a* a*

0.1 mg/l 1.0 mg/l

6

4

b
2

10 5

A* B C b B b C b C b

C b

b

C b

C

0 10

0 25

Mean number of roots per explant

c C
8

a* a*

d D
20

a*

6

15

4

10

b* AB B c A c B c
IAA IBA NAA PAA

b
2

A A B b B* b
0 2,4-D 5

A

AB B b
IAA IBA

B c
2,4-D

0 Control NAA PAA

Control

Figure 4.8: In vitro root formation on four Watsonia species using different auxins at two concentrations. A – W. gladioloides; B – W. lepida; C – W. laccata; D – W. vanderspuyiae. An asterisk (*) denotes significant difference (P < 0.05) between concentrations within a treatment while bars with different letters indicate significant differences between treatments within a species (P < 0.05). Letters in uppercase pertain to treatments at 0.1 mg l-1, and those in lowercase to 1 mg l-1.

In all species, a higher concentration (1 mg l-1) of NAA or 2,4-D significantly decreased plantlet survival ex vitro (Figure 4.9). For the two summer-rainfall species, W. gladioloides and W. lepida, only 2,4-D at the 0.1 mg l-1 level adversely affected

104

Micropropagation

survival, while none of the other auxins were able to improve acclimatization success compared to the control. Interestingly, acclimatization of W. laccata plantlets was significantly reduced in all explants treated with auxins at the higher concentration of 1 mg l-1. For W. lepida, differences between the two controls suggested inconsistencies between treatments in this species.

120

a

A
A

a AB ab A a a AB B* bc

120 100

b B
A* AB * AB

0.1 mg/l 1.0 mg/l

Percent acclimatization

100 80 60 40 20

A*

80 60 40 20 c a B a

A*

a

a

B*

b 0

b

0

120

c C
A A* A* A* A*

A*

120 100 80 60

d D
A a

A*

A a A*

a A

A*

Percent acclimatization

100 80 60 a 40 20 0 Control

ab

a ab ab b b 40 20 0 IAA IBA NAA PAA 2,4-D Control IAA IBA NAA PAA bc c 2,4-D

Figure 4.9: Effect of auxins on ex vitro acclimatization of in vitro-rooted shoot explants. A – Watsonia gladioloides; B – W. lepida; C – W. laccata; D – W. vanderspuyiae. An asterisk (*) denotes significant difference (P < 0.05) between concentrations within a treatment while bars with different letters indicate significant differences between treatments within a species (P < 0.05). Letters in uppercase pertain to treatments at 0.1 mg l-1, and those in lowercase to 1 mg l1

.

If rooting is performed ex vitro, then a short pulse treatment with a high concentration of a stable (slowly metabolised) auxin (commonly IBA) is typically used. Auxin is only required for the induction phase of root initiation, where dedifferentiated cells respond to the applied auxin and begin differentiating into the root meristem (DE KLERK, 2002). High concentrations of auxin present after this stage can be inhibitory to root growth, and may also promote ethylene formation. Although ethylene promoted root
105

Micropropagation

formation when present during the dedifferentiation phase (DE KLERK, 2002), it inhibited root formation during the later induction phase (KALEV and ALONI, 1999). In the in vitro environment however, uptake of auxins occurs over an extended period of time as the explant is always in contact with the auxin-containing medium, and thus use of a stable auxin would therefore be detrimental. Consequently, the preferred choice for root induction in vitro is a less stable auxin (more rapidly metabolised) such as IAA. Other advantages of using IAA include a reduction in callus formation, increased root growth and improved shoot health (DE KLERK, TER BRUGGE and MARINOVA, 1997).

In our studies, IAA, IBA and NAA consistently increased number of roots on in vitro grown shoots across the four Watsonia species studied, while addition of PAA or 2,4D did not influence rooting compared to the control. For three of the species, W. gladioloides, W. lepida and W. vanderspuyiae, an increase in the IBA level from 0.1 to 1 mg l-1 decreased root production. This could result from the slower metabolism of IBA compared to the more rapidly metabolised (and hence less stable) IAA, and so would have a greater effect during a continuous root-induction treatment (DE KLERK, 2002). Both NAA and IAA also increased the number of roots formed, but optimum results were always at the higher concentration of 1 mg l-1. This indicated that these auxins may be more rapidly metabolised (either by conjugation or oxidation) than IBA. It had been speculated that auxins function as growth decelerators rather than growth accelerators in roots (TANIMOTO, 2005). This can, in part, be attributed to the inhibitory effect of ethylene produced by IAA, since treatment with the ethylene action inhibitors, silver nitrate and cobalt chloride, improved rooting in Vicia faba (KHALAFALLA and HATTORI, 2000). Interestingly, a combination of IAA and IBA increased rooting in Ailanthus triphysa cultures (NATESHA and VIJAYAKUMAR, 2004), but combining auxins decreased rooting in Stackhousia tryonii, indicating antagonistic effects (BHATIA, BHATIA and ASHWATH, 2002). However, care needs to be taken when interpreting these results, since both the auxins and the silver nitrate were present throughout the duration of the rooting process, and not only during the defined rooting phases as outlined by DE KLERK (2002).

Although the type and level of auxin were both important for root initiation in all species, the level of auxin applied to Watsonia shoots to stimulate rooting was the major factor affecting plantlet survival ex vitro. In all species, survival of plantlets
106

Micropropagation

treated with NAA or 2,4-D was higher at a concentration of 0.1 mg l-1 compared to plantlets rooted on 1 mg l-1. Except for W. lepida, survival of all species treated with an auxin at 0.1 mg l-1 was similar to plants derived from the control. This does not appear to be related to the number of roots produced, as IAA and NAA both produced high numbers of roots, but plantlet survival was lower in NAA-treated shoots. In contrast, the ex vitro performance of apple ‘Jork 9’ microcuttings was related to the number of roots formed during the in vitro rooting stage (DE KLERK, 2000). W. laccata is of interest because for all auxins tested, plantlet survival was always decreased when the level of applied auxin was 1 mg l-1. This consistent response indicates that acclimatization in this species is dependent on the level rather than the type of auxin. In Thapsia garganica, improved ventilation of culture vessels by using lids with holes plugged with cotton wool improved acclimatization (MAKUNGA, JÄGER and VAN STADEN, 2006), while in Aloe polyphylla cytokinin type was found to affect plantlet survival where BA had a negative impact compared to zeatin and meta-topolin (BAIRU, STIRK, DOLEZAL and VAN STADEN, 2007). Although these factors were not considered in the present study, plantlet survival was satisfactory, even in the controls, and thus there was no need for additional treatments for improvement.

107

Micropropagation

4.4 •

SUMMARY

Establishing effective and efficient procedures for micropropagating plants is an important first stage in the framework of introducing new ornamental plants. Not only does it provide a rapid means for obtaining disease free clones, but provides a foundation for further biotechnological development such as genetic modification that rely on techniques for plantlet regeneration.

• •

For the four Watsonia species examined here, micropropagation protocols were forged through examining hormonal effects on growth and development. Shoot induction from hypocotyl explants required both an auxin and a cytokinin in the medium, while for adventitious shoot multiplication in a solid medium, cytokinin alone was sufficient.

• •

Shoots cultured in a liquid shake medium exhibited greater growth index and multiplication rates compared to those grown on solid media. Effective in vitro rooting was dependent on the type and concentration of the particular auxin applied, and this in part determined the success of ex vitro acclimatisation.

108


				
DOCUMENT INFO
Shared By:
Tags: Chapter
Stats:
views:148
posted:12/15/2009
language:English
pages:20
Description: Chapter 4