Physiological state and clonal variability effects on low temperature storage of in vitro shoot cultures of elms (Ulmus sp.)

Scientia Horticulturae, 56 ( 1993 ) 51-59

51

Elsevier Science Publishers B.V., Amsterdam

Physiological state and clonal variability effects on low temperature storage of in vitro shoot cultures of elms ( Ulmus sp. ) N. Dorion*, B. Godin, C. Bigot Laboratoire de Physiologic V~g~taleAppliqu~e, Ecole Nationale Sup~rieure d'Horticulture, 4 rue Hardy, R.P. 914, 78009 Versailles Cedex, France (Accepted 19 February 1993)

Abstract

As a means of protecting elms against Dutch Elm Disease (DED), in vitro storage of shoot cultures of some elm genotypes under reduced temperatures was investigated. At 7 °C under 8 h of fluorescent lighting, survival rate remained high ( > 82%) during the storage periods (24-30 months), provided the shoots used for the storage were already rooted or about to root ( 1-2 months). Necrosis of some axiUary ramifications at the end of the storage period reduced the multiplication rate from 3-4 to 1. According to the clone, explants taken from the stored plantlets were not as easy to root as the control (20-30% for U. campestris, OCBi ). In order to store elm clones free of DED and to reduce in vitro transfers, an optimal storage period of 18-24 months could be retained. It may be possible to extend the period by improving rooting and growth during the pre-culture phase. Key words: Ulmus; Elms; Cold storage; Micropropagation Abbreviations: DED, Dutch Elm Disease; IBA, indolebutyric acid; PAR, photosynthetically active radiation

Introduction The North American and Eurasian strains of Ophiostoma novo-ulmi (Brasier, 1991 ) cause the death of European and American elm species that are all susceptible to Dutch Elm Disease. Germplasm preservation is therefore imperative. For this purpose, in vitro culture which prevents disease contamination is a useful technique. The micropropagation methods set up for elm clones (Dorion et at., 1987 ) may be used, but serial transfers (every 2 months ) are costly, time consuming and increase the risk of somaclonal variability and loss through contamination. The easiest way of limiting transfer number is to *Corresponding author.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4238/93/$06.00

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~¢. Dorion et aL / Scientia Horticulturae 56 (1993) 51-59

reduce explant growth using cold storage at temperatures ranging from 0°C to 15 ° C. This simple and cheap method has been applied successfully for up to 2 years, to more than 12 genera belonging mainly to fruit trees (Malus, Pyrus, Prunus) and to some evergreen forest trees, especially conifers and eucalyptus (Aitken-Christie and Singh, 1987). The present paper reports on the in vitro medium-term storage of several elm clones. The effects of developmental stage on plant survival and growth is investigated since the physiological state of the shoots as well as the clonal differences determine, to a great extent, the success of a cold storage programme (Aitken-Christie and Singh, 1987). Materials and methods

Five European elm clones were used, two Ulmus carpinifolia (OCBi and OCBa), two hybrids (OMBe m and OMBe r), and one Dutch hybrid ('Commelin'). OCBi and OCBa were introduced in vitro from outdoor shoot sprouts, 'Commelin' from nodal segments of greenhouse grown plants, OMBe m and OMBe r from seed germinated in vitro. Explants were surface disinfected by immersing for 30 s in 70% ethanol then for 20-30 min in 5-10% calcium hypochlorite (according to the material), followed by three rinses in sterile distilled water (Dorion et al., 1987). They were micropropagated in vitro, for several years, from apical and nodal segments as described previously (Dorion et al., 1987). Twenty-four explants per treatment were subcultured in test tubes (200 m m ) containing 15 ml of culture medium and closed with a polycarbonate cap (Bellco). No sealant was applied. The propagation medium contained modified Murashige and Skoog's macronutrients ( 1962 ), (for the composition see Table 1 ). The pH of the medium was adjusted with 1N KOH to 5.5 before autoclaving at 115 °C for 20 min. Explants were stored, for up to 30 months, at 7 °C under fluorescent lights (8 h; 7 Wm-2 PAR provided by a mixture of Sylvania grolux, Philips day light, and Durotest true-lite fluorescent lamps). Before storage ( 1 week, 1 or 2 months) and after preservation periods, explants were kept in a growth chamber at 25 ° C ( d a y ) / 2 2 ° C (night) under 16 h of fluorescent lighting (21 Wm -2 PAR). The explant development was noted at the beginning and at the end of the storage period, then after 15 days or 1 month in the growth chamber. Results were expressed by the leaf number, the rooting percentage, the survival percentage (percent of plants showing at least one regrowing apex ), the percent of branched out plantlets and the multiplication rate (number of new explants, node or apex, recorded from one initial explant). Viability of the plantlets was assessed at the end of a normal propagation cycle of 2 months, on the basis of survival percent, rooting percent, number of nodes or leaves per plantlet and compared with non-stored control plants.

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Table i Culture medium composition Macron utrients (mg 1-9 from Murashige and Skoog (1962) MgSO4,7H20 92.5 CaCI2,2H20 110 KNO3 475 NH4NO3 412.5 KH2PO4 42.5 Micronutrients (mg l - 9 from Heller (1953) MnSO4,H20 0.076 KI 0.01 NiCI2,6H20 0.03 ZnSO4,7H20 1.00 CuSO4,5H20 0.003 H3BO3 1.00 AICI3 0.03 Fe EDTA (mgl -I ) FeSO4,7H20 Na2 EDTA

27.85 37.25

Vitamin mixture (rag l - 9from Morel and Wetmore (1951) Ca panthotenate 1 Inositol 100 Biotin 0.01 Nicotinic acid 1 Pyridoxine 1 Thiamine 1 Sucrose (mM) Activated charcoal (%) (Merck) Agar (Biofit) (%) IBA (/.tM)

30 0.2 0.55 2.5

As often as possible data are given with the standard error (se = _+ t.tr/x/N; t from Student's test at P = 0.05; N is the number of replicates). Results

Influence of storage stage. - Shoots of OMBe r were shifted to low temperature storage conditions 1 week (T1), 1 month (T2) or 2 months (T3) after the last subculture on the propagation medium. Two storage periods of 6 and 18 months were tested. At the beginning of the storage period, developmental stages for the three treatments were as follow: ( 1 ) 7% of the T 1 shoots were rooted but no apical

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N. Dorion et aL / Scientia Horticulturae 56 (1993) 51-59

Table 2 Effect of prestorage culturing and length of the cold storage period on survival of OMBe r plantlets after the first propagation cycle (2 months) No. storage

Storage period 6 months

Survival(%) Number of leaves per plantlet Rooting (%)

74 6.1 _+0.62 100

18 months

T11

T2

T3

T2

T3

74

96

97

87

73

6.8+_ 1.2 78

6.7_+0.8 86

6.6+_0.8 _3

5.2+0.8 78

6.1 +-0.8 _3

iperiod of culturing before the beginning of storage T 1 = 1 week, T2 = 1 month, T3 = 2 months. 2Standard error, P = 0.05. 3Not recorded in this experiment.

growth was observed; (2) 40% of the T2 shoots were rooted and all the shoots were growing, bearing 3.7 + 0.5 leaves; (3) 100% of the T3 shoots were rooted, and bore 6.1 -L-_0.5 leaves. Under the cold storage conditions, shoots continued their development. Rooting was maximum after 6 months (82% for T 1; 100% for T2 ). The number of leaves per shoot increased, reaching maximum values after 18 months for plantlets of the categories T2 ( 12.6 + 1.2) and T3 ( 12.3 + 1.5 ). Under these conditions all plantlets remained alive even after 18 months of storage. Micropropagation was possible, the multiplication rate reached 3.4 and 2.8, respectively, for T2 and T3. However, in the T1 treatment, in spite of normal rooting after 6 months of storage, apical growth did not take place. It resumed only in the growth chamber. Six to eight leaves were produced within 1 month of retrieval from cold storage. In this set of shoots, survival dropped from 100% to 25% between 6 and 18 months of storage. Thus, rooted plantlets are better than unrooted shoots for medium-term preservation of these clones. After one micropropagation cycle, explants from stored plantlets behaved as the non-stored control (Table 2) except for the rooting percent that was somewhat lower than the control. Effect o f shoot batches. - Two batches of explants from OCBi were prepared

for cold storage from the same stockplants after a 10-month interval of normal transfers. Shoots which were subcultured for 1 month were cold stored for 3, 6, 9 and 12 months for the first batch (B 1 ) and 6, 12, 18 and 30 months for the second (B2). At the beginning of the storage period, the growth capacity of each batch was quite different, higher for B1 (7.5___0.6 leaves per shoot) than for B2

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Iv. Dorion et al. / Scientia Horticulturae 56 (1993) 51- 59

coming necrotic. However the survival was always high, since at least one axillary bud remained growing (Table 3 ). At the end of the first propagation cycle the plantlets behaved similarly to the control (survival > 80% and number of leaves per shoot between 7.3 _ 0.5 and 6.5_ 0.5) except for the rooting capacity which decreased between the Months 12 and 18 from 80 ___10% to 30%. It must be noted that this effect was not so evident for OMBe r. C l o n a l e f f e c t . - To study the effect of this factor, four clones (OMBe m and OMBe r, OCBa, and 'Commelin') subcultured for 1 month were stored for 12 and 24 months and compared with the storage behaviour of OCBi. It was shown previously (Fig. 1 ) that OCBi shoots continued to grow beyond 12 Table 4 Influence of cold storage period on vegetative growth and multiplication rate of four elm clones Clones

Storage period (year)

N u m b e r of leaves per s h o o t ~

rate J

OMBe r

0 l 2

4.1 _+0.82 9.5-+1.9 10.1 + 1.5

2.4 1.7

OMBe m

0 1 2

4.2+0.5 9.7+1.3 9.3_+2.3

3.3 1.1

OCBa

0 1 2

9 . 4 + 1.0 13.9_+2.6 14.7_+ 1.3

3.1 1.4

'Commelin'

0

4.7_+0.5 10.9+ 1.5 10.5+2.1

-

1 2

Multiplication

3.1 3.1

IAt the end of the storage period. 2Standard error, P = 0.05. Table 5 Influence of cold storage periods on rooting capacity (%) o f elm clones at the end of the first micropropagation cycle (2 months) Storage

Clone

period

Not stored 1 year 2 years

OMBe r

OMBe m

OCBa

'Commelin'

OCBi

100 88 42

100 97 83

95 95 53

100 94 64

80 95 21

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months in storage. For the clones used in the present experiment, shoot growth stopped before the end of the first year; the number of leaves per plantlet did not increase further (Table 4). Whatever the done, 4-6 leaves were produced during the storage period, which is less than for OCBi (Fig. l ). The final number varied with the donal characteristics, especially its growth capacity during the pre-storage phase (Table 4). After one year, almost 50% of the plantlets were branched similar to the B 1-batch of OCBi. Growth resumed in the growth chamber. After 2 years at low temperature, 100% of the plantlets had produced axillary shoots. This process compensated for apex necrosis, keeping the survival percentage ( 82100%) stable. The propagation capacity of the clones after one year was similar to that of the B2 batch of OCBi, since the number of leaves at this time was quite similar (Fig. l, Tables 3 and 4). It decreased thereafter (Table 4) when part of the axillary shoots became necrotic, except for the dutch hybrid 'Commelin'. Nevertheless it remained at least equal to one, ensuring an exact maintenance of the stock plant. At the end of the first propagation cycle (2 months), the number of leaves per shoot was not different from the non-stored control (6-7 for OMBe m, and OMBe r and OCBi, 8-9 for 'Commelin', and 13-16 for OCBa). The rooting percentage was not affected until one year of storage, but decreased after (Table 5 ). OMBe m seemed to be the least and OCBi the most susceptible (Table 3 ). It must be noted that this clone was also the most difficult to root. Discussion and conclusion

Survival percentage, estimated as 'the demonstration of green terminal parts of the shoots' is always used as a criterion for evaluating the efficiency of preservation methods (Williams and Taji, 1987; Moriguchi et al., 1990). On this basis, it has been shown that elm shoots could survive, on a propagation medium, at 7 °C under 8 h of fluorescent lighting all through the preservation period tested: 24 months for all the clones; up to 30 months for OCBi. Usually, short or medium-term storage of woody plants performed better in darkness at very low temperatures ( 1-4 °C ) as shown for Malus (Lundergan and Janick, 1979 ), Pyrus (Moriguchi et al., 1990) and rose plants (Dorion et al., 1991 ). In our experimental conditions, minimal growth is ensured. Branching from axillary buds took place during the storage period and could relay senescent terminal parts of the shoot. Although in vitro shoots were often successfully stored without roots (Wanas et al., 1986; Aitken-Christie and Singh, 1987; Moriguchi et al., 1990), high level of elm shoot survival is only obtained when storage begins with rooted shoots or shoots ready to root. Sim-

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N. Dorion et aL / Scientia Horticulturae 56 (1993) 51-59

ilar results were reported, for carnation (van Hoof, 1980) and grapes (Barlass and Skene, 1983 ). This may be explained, for elm shoots, according to the micropropagation pattern in which rooting is needed before shoots begin to grow (Dorion et al., 1987). Therefore, it is likely that a better knowledge of the rooting pattern of each clone will lead to the optimisation of the preculture phase and consequently to better long-term preservation. This view is supported bythe differences observed between the two batches of OCBi shoots. B l, which presented the better growth at the beginning of storage, also had the better potentiality to resume micropropagation after one year. Since B2 supported at least six subcultures more than B1 (l year of micropropagation), decreasing growth capacity should be attributable to in vitro culture. The need for reliable storage methods, which also limit in vitro transfer is thus emphasized. To have plantlets usable for efficient micropropagation at the end of the storage period, two other criteria have to be considered. ( 1 ) The multiplication rate at the end of the storage period must be at least similar to the nonstored control. This is the case for all the clones up to 12 months, 18 months for OCBi and 24 months for 'Commelin'. (2) The rooting capacity of the explants arising from the stored plantlets must have the same rooting capacity as that of control explants. In our experiments it decreased after 12 months according to the clones. The phenomenon is particularly evident for OCBi which is also the most difficult to root. Although survival is maintained ( > 82%) until Month 30, the optimal storage period must not exceed 18-24 months according to the clones. Nevertheless, this preservation method could be used efficiently to store elm clones free of DED and to reduce in vitro transfers by 9-12. Improvement of the preservation method needs further research concerning rooting and growth in vitro to establish, for each clone, the duration of the pre-culture phase.

Acknowledgments The authors would like to thank INRA (Institut National Recherche Agronomique ) and MRT (Minist6re Recherche et Technologie ) for financial support. We also thank Professor P. Neumann for reviewing the manuscript and A. Dupond for typing the text.

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