Micropropagation of Cynodon dactylon from leaf and nodal segments

South AfnC811 JO!Jmal of Botany 200 1 67 250-257 Pnnted In South Allies - All nghts reserved

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SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254-629 9

Micropropagation of Cynodon dactylon from leaf and nodal segments S Ramgareeb', MP Watt and AJ Cooke School of Life and Environmental Sciences, George Campbell Building, Universily of Natal, Durban 4041 , South Africa * Corresponding author, e-mail: [email protected]

Received 19 May 2000, accepted in revised form 25 July 2000

Micropropagation protocofs were established for the grass species C. dactylon (L.) Pers. The organogenesis approach involved the culture of nodal segments on MS, 3% (w/v) sucrose and 1% (w/v) agar, and yielded one plant/explant in 10 days. The somatic embryogenesis method utilised the basal segment of young leaves

which were stimulated to produce embryogenic callus on MS, 3% (wlv) sucrose, 1% (w/v) agar and 3mg I·' 2,4-0 in the dark. Somatic embryos germinated (100%) on a hormone-free MS nutrient medium after a 3h laminar air flow drying treatment. This resulted in the production of 40 plantslexplant in 14 weeks.

Introduction Cynodon dactylon (L.) Pers. (Bermuda grass) is an important pasture grass that provides a good ground cover for preventing soil erosion (Gibbs Russell et al. 1990, van Oudtshoorn 1992). It is one of the most widely distributed grass species in the world and displays extensive genetiC diversity by growing in most soil types (Tainton et al. 1976; Gibbs Russell et al. 1990). It is found predominantly in disturbed areas, over-grazed fields , fallow land, road verges and river embankments and grows particularly well in sandy soils, even surviving burial by loose soil (Van Oudtshoorn 1992). Grasses are propagated conventionally either sexually by seeds or vegetatively through nodal cuttings (Ahloowalia 19a4, Greene et a/. 1992). In addition, it is now possible to micropropagate some forage and grass species via either the organogenesis or somatic embryogenic routes of plantlet regeneration. Examples of species for which organogenesis protocols have been published include Poa pratensis (Pieper and Smith 1988), Bambusa glaucescens (Jullien and Tran Thanh Van 1994) and Panicurn virga tum (Alexandrova et al. 1996). Cynodon dactylon (Ahn et al. 19a7, Artunduaga et a/. 19a9), Penniseturn purpureum (Chandler and Vasil 19a4), Panicum virgatum (Denchev and Cong er 1995) and Echinochloa colona (Samantaray et a/. 1997) have all been micropropagated through the somatic embryogenesis morphogenic pathway. As discussed by those authors, the micropropagation approach can allow fo r the more efficient and productive clonal propagation of selected individuals than conventional propagation procedures, particularly when only a single parent plant is available, since very little explant material is required for plant regeneration. Further, in the case of studies aimed at screening individual plants for genetic tolerance to a particular environmental

factor (e.g . saline soils or heavy metal contaminated soils) then the combination of in vitro micropropagation (cloning) and in vitro screening for tolerance to the particular environmental stress would seem to offer the significant advantage that the screening and the controlled multiplication of any desired genotype can be achieved at the same time: This strategy of combin ed screening and micropropagation of desired genotypes has been reported for the selection of plants tolerant to saline cond itions (Patnaik and Debala 1997), copper (Gori et a/. 1998) and chrom ium and nickel (Samantaray et al. 1999). The present study aimed at producing an in vitro micropropagation protocol wh ich could be developed into a system fo r the screening, selection and cloning of individual plants of C. dactylon which would be able to grow in low pH , high aluminium soil con ditions associated with mining wastes. As many mining sites and substrates are prone to wind and water erosion, often the best means of stabilising them is by establishing a vegetation cover (Johnson et al. 1994). Adapted planl populations of C. dactylon may provide one way of developing such a revegetation programme. To date, the somatic embryogenesis micropropagation protocols available for C. dactylon employ young inflorescences as th e explant (Ahn et a/. 19a7, Artunduaga et al. 1989) , but because of the wider applicability (e.g . collection of sterile field material) it was decided to investigate the possibility of establishing a high-yielding in vitro protocol for plantl et regeneration using vegetative material as the explant. For this reason. in the study reported here, two approaches were investigated, viz, shoot and root production from nodal segments and the culture of young leaf tissue for planllet production through somatic embryogenesis.

South African Journal of Botany 2001 . 67 250-257

Materials and Methods Plant Material and Growth Conditions Cynodon dactylon parent plants were grown initiatly from commercial seed in soil in plastic pots (3000cm l) and main-

tained in the greenhouse (25°C day/1SoC nighl). A single parent plant was selected and propagated in the greenhouse through macrocuttings, each comprising a node with axillary buds and root primordia. These cuttings grew into mature plants after two months . The plants were grown in

soil and watered daily. Fertiliser (N:P:K - 2:3:2; 6.3% N, w/v) was applied (1% w/v) once every two months. The grasses were also sprayed once every six weeks with an aphid insecticide (1ml I·' ) (Metasystox R, Bayer, Germany)

251

select the most appropriate carbon source, viz. 3 and 6% (w/v) sucrose, 1.5% (w/v) each of sucrose and sorbitol, 3% (w/v) sorbitol , and a combinalion of 2.3% (w/v) sucrose and 0.1% (w/v) each of ribose, xylose, arabinose, glucose, mannose, galactose and fructose . The most effective auxin type and concentration were selected in a similar manner, on MS nutrient medium. Auxins tested were 2,4-0 (2,4-dichlorophenoxyacetic acid " 3 and 5mg I '); 3mg I ' picloram (4-amino3,5,6-trichloropicolinic acid) and 1.5mg I·' 2,4-0 and 1.5mg I 'picloram. All media contained 1% (wi v) agar and Ihe pH was between 5.6 and 5.S prior to auloclaving. The explants were placed on callus induction media (six explants/30ml in a 95mm Pelri diSh), in the dark at 25°C for 6 weeks, with one subculture on the same medium after 2 weeks.

during the summer months.

Plant regeneration Atter the callus induction slage, Ihe compact embryogenic

Organogenesis studies

call us regions we re separated from those that were soft, watery and non embryogenic and transferred to several

Stolon nodal segments, consisling of a node with axillary buds and 10mm of the internode above and below the node, were excised and the sheath was removed to expose the

buds . They were sterilised for 20 minutes in 1% (v/v) sodium hypochlorite containing a few drops of Tween 20. Explants were cultured on MS salts and vitamins (Murashige and Skoog 1962) and 3% (wlv) sucrose for 10 days. Alternatively, explants were placed on shoot multiplication media consisting of MS salts, White (1943) vitamins , 3% (wlv) sucrose, 0.22g I·' CaC I" 0.014g I·' FeSO" 1mg I·' citric acid, 1mg 1. 1 ascorbic acid and combinations of kinetin (6-fur-

furylaminopurine) (1.5, 3 and 5mg I·' ) and IBA (indole butyric acid) (1 and 3mg I ') for 10 days. The shoots were rooted on MS salts and vitamins, 3% (wlv) sucrose, and 2mg I·' IBA. All media contained 1% (wlv) agar and the pH was 5.6- 5.S prior to autociaving. Other cull ure condilions we re 1 explanV10ml of medium in a 20 x 90 mm glass tube which was maintained in the growth room

(16h lightiSh dark photoperiod, 25°C day/20°C night and 200~E m ' s ').

regeneration media. These all contained MS, 3% (wlv) sucrose, 1% (wlv) agar and various ad ditives, viz. 1% (w/v)

activated charcoal, 12mg I"' ABA (abscisic acid) and 40g I·' PEG 6000 (polyethyleneglycol) , pH 5.6-5.S. Some calli were subjected to a drying treatment, which involved leaving the uncovered Petri dishes containing embryogenic calli on cul-

tu re media, under a laminar flow stream (250 Pal for " 3 or 6h. The calli were then placed on MS, 3% (w/v) sucrose and 1% (w/v) agar. Media were dispensed into 95mm Petri dishes and cultures (4 call i pieces/dish) we re maintained in the growth room (16h lighVSh dark photoperiod, 25°C day/1SoC night and 200 ~E m·' s·'). The treatments were evaluated by recording the number of calli that contained germinating embryos and by counting the germinating embryos in single callus clum ps of known fresh mass, using a Wildt dissecting stereomicroscope.

Hardening-off Plantl ets regenerated through both organogenesis and somatic embryogenesis were acclimatised to greenhouse conditions. The agar medium was washed from the roots

Explant preparation Tillers were harvested from the mature parent plants and

and plantlets were potted into moist soil in plastiC pots (200cm'), enclosed in polythene bags, and placed in the same greenhouse as parent plants (25°C day/1SoC nighl). The bags were open ed daily for increasingly longer periods

kept moist in a container filled with tap water until required.

for one week , after which they were removed permanently.

Somatic embryogenesis studies

The leaf sheath was unfurled and the outer leaves peeled away to obtain the three youngest leaves. During sample preparalion the explants were kept temporarily in half strength MS and 0.1 % (w/v) sucrose. Whole leaves we re sterilised for 15 minutes in 1% (vlv) sodium hypochlorite containing a few drops of Tween 20 and cut into four regions

as shown in Table 3. In later studies, only region 0 (10mm long) of the youngest leaf (leaf 1) (Table 3) was used as the explant. Callus induction Several basal nutrient formulations (Murashige and Skoog 1962, Gamborg et al. 1965, Schenk and Hi ldebrandt 1972, Chu et al. 1975) were tested initially. Thereatter, the Murashige and Skoog (1962) nutrient medium was used to

Plants produced via nodal cuttings were harvested atter 10 days, 1 month or 3 months of growth in the greenhouse . The relative growth rates [(Mass, - Masso)/Mass<> = g g ' month·') of in vitro-produced and macropropagated plants were calculated using the dry mass of each plant type. The dry mass data, atter 1 month growth in the greenhouse, were used to calculate the relative growth rates aher 3 months (i.e. Masso = month 1 dry mass). Results and Discussion

Micropropagation through node culture Shoots and roots developed almosl simultaneously from

Ramgareeb. Watt and Cooke

252

nodal segments of C. dacty/on with in 10 days of culture initiation on hormone ~ free MS nutrient medium, but the yield of plantlel production was very low (1 plant let/explant) (Figure 1. Table 1). Although all explants produced shoals. only 36% of nodes produced two to three shoots, and rooting efficiency was 77%. In an attempt to increase shoot multiplication , nodes were cultured on IBA and/or KIN con taining-media for 10 days (Table 1). However, as Ihe developed shoots did not root on the multiplication media, it was necessary to transfer

these onlo a rooting medium for a further 10 days. The results indicate that the addition of 5mg I ' KIN had a significantly positive effect on shoot yield but reduced subsequent rooting and. consequently, plantlet yield (Table 1). This contrasts with the findings of Alexandrova et a/. (1996) and Flachsland et a/. (1997) , who reported that rool formation is suppressed on cytokinin containing-media but that rooting usually resumes once the shoot is transfe rred onto a hormone-free MS nutrient medium or MS nutrient media supplemented with a high (8%) sucrose concentration . In spite of other workers having reported greater rooting successes

than in the present sludy. Ihey also experienced low planllet regeneration yields from nodal cuttings of grasses.

Examples include 0.95 plants/explant for Poa pratensis (Pieper and Smith 1988). 0.51 plan Is/explant for Bambusa g/aucescens (Jullien and Tran Thanh Van 1994) and 1.2 shoots/node for Panicum virga tum (Alexandrova et a/. 1996). A high yield. 7.6 shoots/node, was obtained for Setaria anceps in a SAP (6-benzylaminopurine)-containing medium (Flachsland et a/. 1997) but this was achieved in 60 days. six times longer than with the protocol used in Ihe present study. After 100% success with hardening-off the in vitro plantlets , their growth rates in the greenhouse were com -

pared with Ihose of plants produced through macro-cuttings in potting soil, both propagation procedures being initiated

on the same day. Atter ten days. when in vitro- produced planilets were Iransferred to the greenhouse. their dry mass was determined and compared with thai of conventionally produced plants (Table 2). Dry mass determinations were then repeated one and Ihree months later. The results show that, after one month in the greenhouse, there was no significant difference in dry mass between the two types of plants. However. the in vitro-produced plantlels had a higher relative growth rate (182.2g g.' month·') than traditionally grown plants (36.8g g ' month') considering their considerably lower initial mass. Between one and three months the

plants had similar relalive growth rates (Table 2). Therefore, even though in vitro-produced plants were initially (atter 10 days) five times smaller than Ihose traditionally grown. this did nol negatively affect their subsequent growth in the ex vitro environment.

Figure 1: Plantie! regenerated from nodal segment via organogen-

esis . Shoot and root development was acheived in 10 days on media containing MS nutrients, 3% sucrose and 1% agar. Magnification 1.5X

Micropr opag ation thr ough somatic embry ogenesis The process of somatic embryogenesis in C. dacty/on When basal segments from young C. dacty/on leaves were

Table 1: The effect of plant growth regulators on shoot and rool proliferation from nodal segments. The media also contained MS , White (1943) Vitamins, 0 .229 I ' CaCb, 0 .01491 ' FeSQ., lmg I ' citric acid, lmg I' ascorbic acid , 3% sucrose and 1% agar. The rooting medium included MS , 3% sucrose. 1% agar and 2mg I ' IBA . . = in this hormone-free MS nutrient treatment , shoot and root proliferation were achieved in a single medium . Data were recorded 10 days after culture initiation for shoot proliferation and 20 days after laler for rool induction. n = 20. Mean values for the hormone treatments represented by different alphabetical letters were significantly different {Scheffes multiple range test p < 0.05

Plant hormone (mg I ') none~

1.5 kin + 318A 3 kin + liSA 5 kin

% ex plants with shoots

100b 56a 100b 100b

% explants with > 1 shoot

36b Oa 55bc 64c

% explan ts rooted

Plant yield/explant

77c

1.0 0 0.7 0.6

Oa 46b 36b

253

South African Journal of Botany 200 1. 67 250-2 57

Table 2: A comparitive study on the performance of plants regenerated from nodal segments in vitro and from macrocuttings . Total dry mass was recorded after 10 days , 1 month and 3 months and re lative growth rales (AGR) were calculated after 1 and 3 months. n = 10. a- b as in Table 1 1 month

10 days

dry mass (9) 0.011 3a 0.0533b

propagatory route In

vitro

conventional

dry mass (9) 2.07a 2.03a

cultu red on MS nutrient media suppl emented with 3mg I·' 2,4-0 , callus proliferation was observed after one week incu-

bation in the dark. Embryogenic (compact, opaque and wh ite to pale yellOW, with small dense cells each showing a single prominent nucleus) and non-embryogenic calli (soft, watery and gelatinous, with large. highly vacuolated and usually multinucleate cells) were distinguishable only after tour weeks. Six weeks after culture initiation, fully-formed globular. notched, embryos (Figure 2A, 8), similar to those descri bed by Fischer et al. (1997) for wheal. we re visible on Ihe surface of the compact callus. AI Ihis time the embryogenic calli were transferred to regeneration media. One

week laler, shool primordia and roots (Figure 2C) were visible and seedling establishment occurred in the following six to eight weeks (Figure 20).

RGR (9 g ' month ') 1B2.2b 36.77a

3 months

dry mass (9) 9B.B7a 1OB.91 a

RGR (g g ' month ') 15.6a 17.6a

commonly used tor the micropropagation of grasses, including cereals are MS and 2- 3% sucrose (Krishna raj and Vasi l 1995). Nevertheless, other formulations and sugars have been shown to be superior for some species. For exam ple,

85 (8hattachatya and Sen 1980). SH (Kuklin and Conger 1995) and N6 (Chen et al. 1995) have proved superior for Oryza sativa, Dactylis glomerata and Avena sativa, respectively, and sorbitol tor Zea mays (Swedlund and Lacy 1993). In C. dactylon. MS (Table 4) and 3% sucrose (Table 5) were the most effective in the production of embryogenic call us

(80% explants producing callus). The SH , N6 and 85 media were at least 50% less effective compared with MS (Table 4). Similarly, callus production decreased from 80% to 35% when sucrose concentration was increased from 3 to 6% and by 41% when 2.3% sucrose was combined with simple sugars; no callus production occurred when sorbitol was the

Establishment of the protocol

carbohydrate source (Table 5). The effecl of the supply of

Different parameters were investigated to optimise the

various simple sugars (ribose, xylose, arabinose, glucose, man nose, galactose and fructose) was tested as these are

induclion of embryogenic callus from C. dactylon leaf malerial. These included the source and age of the leaf explant. the nutrient formulation , type and concentration of sugar and auxin in the culture medium. Various studies have shown that young , immature leaves are the most appropriate vegetative explant for somatic

embryogenesis in the Poaceae (Shatters et al. 1994, Chen et at 1995, Samantaray et al. 1997). Consequenlly, in this study, the three most immature leaves in a leaf roll were used initially and referred 10 as leaf 1 (youngest), leaf 2 and

leaf 3 (oldest). Each was divided into four regions , as shown in Table 3, and cu ltured on a medium containing 3mg I·' 2,4O. Callus production was direclly related to leaf age and region , with 80% of explants from the basal end of the youngest leaf (region 0 of leaf 1) producing callus (Table 3) . As microscopic observations indicated that this callus was

highly embryogenic, this explant was used in all subsequent investigations. The nutrient composition and carbohyd rate sources most

Table 3: The effect of leaf age and region on callus induction . Leaves were cultu red on MS + 3% sucrose + 1% agar + 3mg 1-' 2,40. Leaf 1 = youngest leaf, leaf 3 = older outer leaf. Each leaf was diVided into four regions (A, S, C, 0) . Data were recorded 28 days after culture initiation. n = 20 . a-c as in Table 1

% explanls with callus

Leaf region

r

Leaf 1

A

8

C 0

Oa 5a 60b BOc

Leaf 2

Oa 57c Oa 14b

Leaf 3

Oa 11 b Oa Oa

essential for Ihe synlhesis of plant cell walls (Fry 1988) and are also known to increase embryo formation (Cai et al.

1992). However, they proved ineffective for callus induction in the present study (Table 5). In addition to suitable basal nutrient medium and carbohydrate source , the induction of active cell differentiation

requires an auxin (Vasil 1987). Usually, in the culture of grasses, 2,4-0 is the sole plant growth regulator added 10 the medium to induce active cell proliferation (Krishnaraj and

Vasil 1995). However, in Zoysia japonica (Japanese lawngrass) (Asano 1989) and Eleusine coracana (Finger millet) (Eapen and George 1990) picloram was used more effectively than 2.4-0 . Consequently, both were evaluated independently and in combination (Table 6) . The results obtained indicate Ihat, in C. daclylon, 2,4-0 at 3mg I·' induced significantly more callus (80% explants producing callus) than any of the other tested auxin types and combinations. Lowering the concentration or eliminating the auxin from

Table 4: The effect of nutrienl formulations on callus induction from basal leaf segments. MS = Murashige and Skoog 1962; 85 = Gamborg et al. 1968; SH = Schenk and Hildebrandt 1972; N6 = Chu et al. 1975. Other media constituents included 3% sucrose, 1% agar and 3mg I ' 2,4-0 . Data were recorded 28 days after culture initiation . n = 40 . a-c as in Table 1 Nutrient formulation

MS SH

N6 85

% explants with callus

BOc 36b l Ba 26.5b

254

Ramgareeb. Watt and Cooke

Fig ure 2: Somatic embryo development in Cynodon dactyfon. The stages shown are early globular notched (A) , lale globular notched (8 ) and mature (C) . The mature embryos were stim ulated to germ inate afte r a 3h fast-drying treatment and developed into plantlets (0) on MS nutrients. 3% sucrose and 1% agar. Magn ification (A), (8) and (C) = 30x, (0) = 1.5 X

255

South Afncan Journal of Botany 2001, 67 250-257

Table 5: The effect of various types and concentrations of sugars on callus induction from basal leaf segments, T he tested media also included MS + 1% agar + 3mg I ' 2,4-0 , Data were recorded 28 days after culture initiation. n = 40 . a-c as in Table 1

Table 6: The effect of auxin type and concentration on callus induction from basal leaf segments. The callus induction medium also contained MS + 3% sucrose + 1% agar. Data were recorded 28 days after cultu re initiation. n = 40 . a-c as in Table 1

Sugar

Auxin

Type

%

% explants with callus

sucrose sucrase sorbitol sucrose + sorbitol sucrose + ribose + xylose + arabinose + glucose + mannose + galactose + fructose

3

BOc 45b

6

3 1.5 + 1.5

50b

2 .3 + 0.1 each

39b

0.

the culture medium is o~en the only basic requirement for planUet regeneration from grass embryogenic callus (Ahn el al. t 987, Shatters el al. 1994, Denchev and Conger 1995, Krishnaraj and Vasil 1995, Kuklin and Conger 1995). Therefore, calli with developing embryos produced on 3mg I" 2,4-0 were exposed to different regeneration treatments , all

of which excluded 2,4-0 (Table 7). Only 50% of Ihe somatic embryos transferred to MS, with or without 1% activated charcoal , germinated. Abscisic acid, which has been used in the maturation and germination of somatic embryos of

grasses (Chandler and Vasil 1984, Ray and Ghosh 1990), failed to stimulate germination in any of the cultures. In some studies, somalic embryos were stimulated into

germination by reducing the water potential of the culture medium, either by the incorporation of PEG (Siddeswar and Kavi-Kishor 1990) or by using a high sucrose concentration (Cardona and Duncan 1997) in the culture medium. Th e former approach (40g I" PEG) failed to stimulate embryo germination in C. daelylon (Table 7). In other investigations workers have employed physical dehydration treatments, either slow drying (70-90% relative humidity, non-sealed Petri dishes in the laminar flow for 1 day-3 weeks) , (Gray 1987, Park el al. 1994) or fast drying (opened Petri dishes in the laminar flow for 50-120min) (Capuana and Debergh 1997). The slow (21 days) drying method was used by, amongst others, Gray (1987) for orchardgrass and by Park el al. (1994) for the gymnosperm, Pieea glauca, with 4 and 22% germination success, respectively. The fast (90mm) Table 7: The effect of various treatments on the germination of somatic embryos. All treatments included MS + 3% sucrose + 1% agar. Clumps of calli which cu ltured on regeneratIon media contained approximately 200 embryos g" fresh mass. The cu ltures were incubated in the light and observations recorded 2 weeks a fter culture initiation . n = 20 . a-d as in Table 1

%

Treatment

catti with germinating embryos

None 1% activated charcoal 12mg I' ABA

50b

409 I' PEG

0. 40b

drying

1h

3h 6h

45b Oa

100d 70c

Type 2,4-0

% embryo germination SOb 4Bb

0.

Oa 63b 100d 80c

picloram 2,4·0 + picloram

mg I"

% explants with callus

1 3

1.31.

5 3

1.5 + 1.5

80d 52 .9b 46.2b 68c

drying option resulted in 9.2% germination of chestnut

somatic embryos (Capuana and Debergh 1997). In the present invesligation only the fast (180mm) drying method was tested and as this yielded 100% embryo germination (Table 7), no other melhod was_pursued. In conclusion, a protocol has been established for plantlet regeneration through somatic embryogenesis in C. daclylon. It utilises a two-stage cu lture procedure, both with simple cul ture media formulations comprised of MS nutrients, 3% sucrose, 1% agar (for both callus induction and regenera-

tion) and 3mg I" 2,4-0 (for callus induction). These media compositions are similar to those reported for the regeneration of various other grasses and cereals from leafbase sec-

tions (Shatters el a/. 1994, Chen el al. 1995; Denchev and Conger 1995, Krishnaraj and Vasil 1995, Samantaray el al. 1997). In addition, this method involves a 3h drying treatment before transfer to the regeneration medium.

Comparison of propagation protocols with conventional macrocuttings

Th e conventional macrocutting propagation method (details not given here) and the in vitro organogenesis protocol use the same explant type (nodal segments) and both result in one plantletJexplant in 10 days. The somatic embryogenesis protocol employs basal segments from young leaves and resu lts in approximately 200 planUets g" fresh mass (±40 plants/explant) in 14 weeks . Therefore, one may estimate that from a parent plant with 50 nodes , the resulting yields would be relatively similar, i.e. 2 500 pl ants/14 weeks via both macrocuttings and in vitro organogenesis and 2 000 plants/14 weeks through somatic embryogenesis. However, the methods differ in terms of ease of manipulations, labour

time and amount of media reqUired . For the production of 2 500 plants/14 weeks, the macrocuttings and in vitro organogenesis methods each requires the preparation of 2 500 explants, at an estimated 1hl1 00 explants, and 251 of culture media and 5001 of potting soil, respeclively. The somatic embryogenesis method uses approximately 50-50 explants, at an estimated 2.5h/100 explants, and 500ml of media. The purpose of establishing a micropropagation protocol for the grass C. daclylon in this study was for it to be the basis of an experimental protocol to select and produce an

initial batch of cloned individuals with desirable traits which

256

could then be rap idly produced in conventional macropropagation programmes. The in vitro protocols established in the present study are being applied currently to the screening for aluminium-tolerant genotypes of C. dactylon. For this purpose, the macronutrient formulation of the culture media reported here (MS nutrients) was modified using the MINTEQA2 Chemical Speciation Program (Allison et al. 1991) to ensure maximum AP- activity, wi th no negative effects on regeneration (Ramgareeb et al. 1999) . Acknowledgements - We thank EMPR Services (South African Chamber of Mmes), the National Research Foundation and the University or Natal Research Fund for financia l support. We would also like to acknowledge the assistance of P 8erjak and P Maartens with photography and P Berjak for comments on the manuscript.

References Ahloowalia BS (1984) Forage grasses, In: Ammirato PV, Evans OA, Sharp WR, Yamada Y (eds) Handbook of Plant Cell Culture. Crop Species, Macmillan Publishing, New York. pp 91-125 Ahn BJ, Huang FH , King JW (1 987) Regeneration of Bermudagrass cultiva rs and evidence 01 somatic embryogenesis. Crop Science 27:594-597 Alexandrova KS , Denchev PO, Conger BY (1996) Micropropagation of Switchgrass by node culture. Crop Science 36. 1709-1711 Allison JO, Brown OS . Novo-Gradac KJ (1991) MINTEQA2IPROOEFA2: A geochemical assessment model for environmental systems. Version 3.0 User's manual. United States Environmental Protection Agency, Athens, Georgia Artunduaga IR, Talliaferro CM, Johnson SB (1989) Induction and growth of callus from immature in florescences of 'Zebra ' Bermudagrass as affected by casein hydrolysate and 2.4-0 concen tration. In Vitro Cell. Dev. BioI. 25: 753- 756 Asano Y (1989) Somatic embryogenesis and protoplast culture in Japanese lawngrass (Zoysia japonica). Plant Cell Reports 8:

140-141 Bhattacharya P, Sen SK (1980) Potentiality of leaf sheath cells for regeneration of rice (Oryza sativa L.) plants. Theoretical and Applied Genetics 58: 87-90 Cai Q , SzareJko I, Polok K, Maluszynski M (1992) The effect of sugars and growth regulators on embryoid formation and plant regeneration from barley anther culture. Plant Breeding 109:

218-226 Capuana M, Oebergh PC (1997) Improvement of the maturation and germination of horse chestnut somatic embryos. Plant Cell Tissue and Organ Culture 48: 23-29 Cardona CA, Duncan RR (1997) Callus induction and high efficiency plant regeneration via somatic embryogenesis in Paspalum. Crop Science 37: 1297- 1302 Chandler SF, VasillK (1984) Optimization of plant regeneration from long term embryogenic callus cultures of Pennisetum purpureum Schum. (Napier grass). Journal of Plant Physiology 117: 147- 156 Chen H, Xu G, Loschke DC , Tomaska L, Rolfe BG (1995) Efficient callus formation and plant regeneration from leaves of oats (Avena sativa L.) . Plant Cell Reports 14: 393-397 Chu CC , Wang CC , Sun CS, Hsu C, Yin KC , Chu CY. Bi FY (1975) Establishment of an efficient medium for anther culture of rice through co mparative experiments on the nitrogen sources. Scientia Sinica 18: 659-668 Oenchev PO , Conger BV (1995) In Vitro culture of switch -g rass: Influence of 2,4-0 and picloram in com bination with benzyladenine on callus initiation and regeneration. Plant Cell Tissue and Organ Culture 40: 43-48

Ramgareeb. Watt and Cooke

Eapen S. George L (1990) In fluence of phytohormones, carbohydrates, amino acids. growth supplements and antibiotics on somatic embryogenesis from immatu re inflorescence cultures of finger millet. Plant Cell Tissue and Organ Cu lture 22: 87-103 Fischer C, Volker S, Fleig-Eberenz S, Neuhaus G (1997) Induction of zygotic polyembryos in whea t: Influence of auxin (on) polar Iransport. Plant Cell 9: 1767- 1780 Flachsland E, Bovo OA, Mrog inski LA, Tomei C (1997) Micropropagalion of Setaria anceps cv. 'Nandi' (Gramineae) through single-node-stem explanls. Siocell 21 : 187-193 Fry SC (1988) WaJl BiosynthesIs. In: The Growing Plant Cell: Chem ical and metabOlic analysis. Longman Scientific and Technical, London , pp 188-218 Gamborg OL, MiUer RA , Ojima K (1968) Nutrient requ irements of suspension cultures of soybean root cells. Experimental Cell Research 50: 151-1 58 Gibbs-Russell GE, Watson L, Koekemoer M, Smook L, Barker NP. Anderson HM , Oallwitz MJ (1990) Grasses of Southern Africa. Memoirs of Ihe Botanical SUlVey of South Africa. No. 58, 437 pp. National Botanical Gardens, Botanical Research Group, Pretoria Gori p, Schiff S, Santandrea G, BenniciA (1998) Response of in vitro cultures of Nicotiana tabacum L. to copper stress and selection of plants from Cu-tolerant callus. Plant Cell Tissue and Organ Culture 53: 161-169 Gray OJ (1987) Quiescence in monocotyledonous and dicotyledonous somatic embryos induced by dehydration. Hort Science 22:

810-8 14 Greene SB, Lancaster OM , Pee JM. Turpin JM , Eichhorn MM (1992) Propagation of hybrid Bermudagrasses from halVested culm s. Agronomy Journal 84: 31 - 33 Johnson MS. Cooke JA, Stevenson JKW (1994) Revegetation of Metalliferous Wastes and Land after Metal Mining. In: Hester RE , Harri son RM (eds) Mining and its Environmental Impact, Issues in EnVironmental Science and Technology 1: 31-48 Jullien F, Tran Thanh Van K (1994) Micropropagation and embryoid formation from young leaves of 8ambusa glaucescens 'Golden goddess'. Plant Science 98: 199-207 Krishnaraj S , Vasi l IK (1995) Somatic Embryogenesis in Herbaceous Monocots. In: Thorpe TA (ed .) In Vitro Embryogenesis in Plants. Kluwer Academic Publishers , Oordrecht, pp 417-470 Kulkin AI, Conger BV (1995) Enhancemen t of somatic embryogenesis in orchardgrass leaf cultures by epinephrine. Plant CeJJ

Reports 14: 641-<>44 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiolog ia Planterum 15: 473-497 Park YS, Pond SE. Bonga JM (1994) Somatic embryogenesis in white spruce (Picea glauca): genetic control in som atic embryogenesis exposed to storage, maturation treatments, germination, and cryopreservation. Theoretical and Applied Genetics 89 : 742-750 Patnaik J, Debata SK (1997) Regeneration of planUets from NaCI tolerant callus lines of Cymbopogon martinii (Roxb.) Wats. Plant Science 128: 67-74 Pieper MA, Smith MAL (1988) A whofe microculture selection system for Kentucky Bluegrass. Crop Science 28: 611-614 Ramgareeb S, Watt MP, Cooke JA (1999) Assessment of All> availability in callus culture media for screening tolerant genotypes of Cynodon daclylon. Plant Cell Tissue and Organ Culture 56 :

65-68 Ray OS, Ghosh PO (1990) Somatic embryogenesis and plant regeneration from cultured leaf explants of Zea mays. Annals of Botany 66: 497-500 Samantaray S, Rout GR, Das P (1997) Regeneration 01 plants via somatic embryogenes is from leaf base and feaf lip segments of

South African Journal of Botany 200 1, 67 250--257

Echinochfoa colona. Plan! Cell Tissue and Organ Culture 47:

119-1 25 Samantaray S, Rout GR, Das P (1999) Chromium and nickel toler~ ance of Trema orientalis (Blume) L. in tissue culture. Acta Physiologiae Plantarum 21 : 27- 35 Schenk RU , Hildebrandt AC (1972) Medium and tech niques for Induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Canadian Journal of Botany 50: 199-204 Sha tters RG, Wheeler AA, West SH (1994) Somatic emb ryogenesis and plant regeneration from callus cultures of 'Tifton 9' Bahiagrass. Crop SCience 34: 1378-1384 Siddeswar G, Kavi ~ Kishor PB (1989) Plant regeneration from po l y~ ethyleneglycol adapted callus of rice. Current Science 58:

926-928

Ed iled by J van Sladen

257

Swedlund B, Lacy RD (1993) Sorbi tol as the primary carbon source for the growth 01 embryogenic callus of maize. Plan t Physiology

103: 1339- 1346 Tainton NM, Sransby 01 , Booysen PO (1976) Common veld and pasture grasse s of Natal, 92 pp . Shuler and Shooler, Pietermaritzburg Van Oudlshoorn F (1992) Guide to grasses in Southern Africa , 140 pp. Briza, Arcadia Vasil IK (1987) Developing cell and tissue culture systems for the improvement of cereal and grass crops. Journal of Plant

Physiology 128: 193-218 White PR (1943) Nutrient deficiency studies and an Improved inor~ 9anic nutrient for cultivation of excised tomato rools. Growth 7:

53-65