South African Avocado Growers’ Association Yearbook 1987. 10:36-39.

Proceedings of the First World Avocado Congress

 

 

Propagation of avocado rootstocks by tissue culture

 

FERNANDO PLIEGO-ALFARO, CL ENCINA and A BARCELO-MUFIOZ
Centro de Investigacion y Desarrollo Agrario,

Cortijo de la Cruz, Churriana, 29140 Malaga, Spain

 

 

INTRODUCTION

Seedling avocado rootstocks show a great amount of variability in their productivity (Gillispie, 1952; Ben­Ya'acov, 1976), and their tolerance of saline and limestone soils (Kadman, 1968).

 

Tissue culture techniques are widely used to propagate many different species (Murashige, 1978) The capa­city however, for in vitro regeneration of many woody plants, including the avocado, seems to be limited to juve­nile material (Pliego-Alfaro, 1981).

 

This investigation was designed to determine the in vitro morphogenetic capacities of two avocado rootstocks - a Mexican seedling, IV-8, selected because of its high and uniform production over several years; and the GA-13, a selected line shown to be tolerant to saline and limestone condi­tions (Kadman & Ben-Ya'acov, 1980).

 

MATERIALS AND METHODS

The IV-8 rootstock from la Mayora Experimental Station, Malaga, Spain, was selected from an orchard of 225 Mexican seedlings grafted with the Hass cultivar. Production data (kg/tree), productivity (g/cm2 trunk) and tendency to yield variation in successive years (Monselise & Gold­schmidt, 1982) were determined be­tween six and nine years after planting.

 

The budwood from the GA-13 root­stock was kindly provided by the Volcani Center Institute, Bet Dagan, Israel.

 

Shoots growing actively were ob­tained by two methods: (1) after heavy pruning of the selected tree in the field in the case of the IV-8 rootstock and (2) in the case of GA-13 rootstock, by grafting scions onto seedling root­stocks by conventional procedures (Hartmann & Kester, 1981); the plants were then grown under greenhouse conditions. The excised shoots, 15­25 cm in length, with leaves removed, were sterilised by immersion in 0,5 per cent sodium hypochlorite solution for 10 min. Immediately afterwards, they were divided into 1 - 1,5 cm long nodal sections each containing a lateral bud. They were sterilised once more as explained previously, washed three times in autoclaved distilled water and then cultured after carefully cutting away the edges of the nodal sections observed to have been damaged by the disinfectant.

 

After four to six weeks, 1,5 cm long shoots were obtained for the multipli­cation and rooting experiments. Sub­culturing was always carried out at four-weekly intervals and in each experimental group the length of the shoots was between 15 and 20 cm.

 

Shoot multiplication of the IV-8 root­stock was accomplished by using a double-phase medium containing the Murashige & Skoog salt formulation, but with the macro-elements at half strength and in the following concen­trations (mg/L): sucrose (30 000); i­inositol (100); thiamine HCI (0,4); and agar (8 000). The BA-concentration was 0,65 mg/L in the solid phase and 0,1 mg/L in the liquid phase. The final medium developed for shoot multi­plication of the GA-13 rootstock inclu­ded the mixture of macro-elements, N₄₅K (Margara, 1984), the MS micro­elements (Murashige & Skoog, 1962) and the following concentrations (mg/L) of sucrose (30 000); i-inositol (100); thiamine HCI (0,4); benzyladenine (1); and Bacto-agar (8 000). In this medium the shoots needed to be re­cultured once a week. The rooting capacities of the shoots were deter­mined by using a two-step sequence involving three days of culture in a basal medium with MS macro-elements diluted to 0,3X and 1 mg/L supplement of IBA (indole-butyric acid), and then subsequently transferring them to a similar medium without IBA but with 1 mg/L activated charcoal added (Plie­go-Alfaro, 1981).

 

Solid media contained 25 mf of medium per test-tube. When a double layer was used, 3 ml of liquid medium was added on top of the solid phase. All cultures were incubated at 25°C, with a light intensity of 1 500 lux and an 8:16 h dark:light schedule.

 

RESULTS

 

Selection of rootstocks

The IV-8 rootstock was selected be­cause of its low tendency to biennial bearing (I = 0,07), moderately high production (45,6 kg/year) and very high productivity (290 g/cm² trunk) over the four-year data-recording pe­riod.

 

Culture initiation and shoot multiplication

A series of experiments was conducted to test the effects of the MS macro­elements at different strengths (0; 0,25X; 0,5X;1X) on the growth of lateral buds of the Mexican rootstock IV-8. The 0,5X dilution was chosen as standard, since it had given an average shoot length of 1,60 ± 0,15 cm and a low percentage of necrotic buds. The general appearance of the shoots grown at the 1 X level was good, but many buds died. In contrast, cultures of GA-13 rootstock could be initiated at the 1 X level of macro-elements. To maintain shoots in stock, several media were tested with different salt formu­lations: Gamborg (Gamborg et al, 1968); Murashige & Skoog; and three modifications of the latter; N₄₅K, N₃₀K and N₃₀Ca (Margara, 1984). In terms of main shoot length (2,9 ± 0,1 cm); num­ber of axillary shoots per culture (3,7 ± 0,3); and average length of axil­lary shoots (0,7±0,1 cm); no significant differences were observed between the different formulations and the MS mixture used as control. However, it should be noted that the shoots cultured in the N₄₅K medium appeared healthier and had a much higher number of leaves per culture, eg 4,2 ± 1,5 (N₄₅K) against 2,0 ± 0,4 (MS), and consequently this was chosen as the' standard.

 

Preliminary experiments not reported on had shown that the cytokinin BA was superior to kinetin and 2-isopentenyl­adenine in inducing shoot proliferation of avocado tissue cultures, therefore a test with different BA concentrations (0, 0,3, 1 and 3 mg/L) was carried out. At 0,3 mg/L BA, the lateral buds of the Mexican rootstock (IV-8) gave rise to shoots with an average length of 2,1 ± 0,2 cm, a leaf number of 2,1 ± 0,4 and also of better general appearance than the control. At 1 and 3 mg/L BA, however, the shoots were aberrant and succulent and the leaves were pale green in colour. Reculture of these shoots at the 0,3 mg/L BA concen­tration did not induce any axillary proliferation, thus a new set of experi­ments was carried out to test the effects of BA concentrations in the range 0,3 to 1 mg/L. At the 0,65 mg/L BA level a proliferation rate of 2,2 ± 0,4 mg/L axillary shoots per cul­ture was obtained. However, after sub­culturing, many shoots developed api­cal necrosis and the multiplication rates decreased drastically.

 

Shoots of the GA-13 rootstock grew and proliferated well in a basal medium supplemented with 1 mg/L BA, but once more, as in the case of the IV-8 rootstock, apical necrosis was ob­served in the shoots and the initial proliferation rates (2,0 ± 0,60) de­creased on subculturing.

 

The apical necrosis problem was resolved by using a double-phase tech­nique, which included the optimum solid medium developed for each root­stock plus a supplement of liquid medium on top. A range of BA concen­trations in the liquid phase from 0 to 1 mg/L were tested.

 

Cultures of the IV-8 rootstock in double-phase medium showed a multi­plication rate of three to four axillary shoots per culture and apical necrosis disappeared completely. Lack of BA in the liquid phase did not prevent shoot multiplication. However, when present at 0,1 mg/L, a slight increase in axillary shoot lengths was observed and con­sequently this concentration was chosen as the standard for the liquid phase. Unfortunately, vitrification was frequently observed in double-phase cultures and main shoot lengths; the number of axillary shoots per culture and the number of leaves per culture all decreased with subculturing while the percentages of aberrant and vitrified shoots increased (Table 1).

 

 

 

 

 

 

 

 

The responses of shoots of the GA­13 rootstock to double-phase medium was much less than those observed with the IV-8 rootstock material. Main shoot lengths decreased dramatically after three subcultures and most of the shoots appeared aberrant. Attempts to improve the nutrient media conditions by decreasing the concentrations of salts and using different levels of BA and other cytokinins, eg kinetin and zeatin in both phases, failed to give positive results. Consequently a new set of experiments was carried out to test the effectiveness of multiple re­culturing at short intervals as a preven­tive procedure against apical necrosis. Four types of reculture treatments were used in these. experiments, one recul­ture every four weeks (control), one every two weeks, one each week, and three each week. The last two treat­ments gave far superior results in terms of main shoot length, number and length of axillary shoots and absence of apical necrosis. The recorded values for average axillary shoot numbers, average axillary shoot lengths and average main shoot lengths were respectively 4,80 ± 0,33; 1,20 ± 0,11 and 3,05 ± 0,21 for the three recultures per week treatment. For one reculture per week, the respective values were 3,30 ± 0,63; 0,89 ± 0,11 and 2,72 ± 0,20. Since no necrotic tissues were ob­served after any of these treatments, practical considerations led to the one reculture per week chosen as the standard.

 

The rooting capacities of the shoots obtained from the double-phase stock of the IV-8 rootstock, or from the multiple recultured single-phase stock shoots of the GA-13 rootstock, were examined following the two-step proce­dure of Pliego-Alfaro (1981) for mature avocado material. Thirty per cent of the shoots from the IV-8 rootstock rooted in comparison with only 5 per cent of the GA-13 rootstock shoots.

 

DISCUSSION

A selection of avocado seedling root­stocks grafted with the Hass cultivar was made. The criteria were high pro­duction and productivity, and a low tendency to biennial bearing. A great amount of variability among individual trees was found, in agreement with the reports of other authors (Gillespie, 1952; Ben-Ya'acov, 1976).

 

The budwood for culture initiation of a selected rootstock (IV-8), was ob­tained after heavy pruning of the tree. This technique has been successfully used to induce rejuvenation in other woody plants normally difficult to propagate in vitro (Franclet, 1979). The morphogenetic in vitro capacity of another rootstock, GA-13, which had been selected in Israel for its high tolerance to saline and limestone soils (Kadman & Ben-Ya'acov, 1980), was also studied.

 

Genotypes play an important role in the subsequent in vitro behaviour. In the case of the IV-8 rootstock, the MS macro-elements had to be reduced to half strength, while a modified MS mixture, N₄₅K (Magara, 1984), potassium enriched and nitrogen reduced, proved to be more suitable to the GA­13 rootstock. Anderson (1984) reports growth improvement in some rhodo­dendron cultivars after reducing the concentration of macro-elements in the MS formulation.

 

The BA requirements for each root­stock were also different: although the 1 mg/L concentration chosen for shoot multiplication of GA-13 was toxic for IV­8, a reduction to 0,65 mg/L proved to be satisfactory. Nevertheless, it was im­possible to maintain the shoots in stock at their optimum BA levels for both root­stocks, because apical necrosis was observed in most of the cultures.

 

In a previous work, Pliego-Alfaro (1981) reports an improvement in shoot quality after applying solutions con­taining BA on top of the solid medium. More recently, Molnar (1985) and Viseur (1985) report significant in­creases in the multiplication rates of many different genera, by using a liquid phase including mineral salts, sugar, vitamins and cytokinins on top of the solid phase. This technique gave different results in the two rootstocks. Shoots of the IV-8 rootstock proliferated quite well and although a progressive degeneration of shoot quality with subculturing was observed, at least 50 per cent of the cultures appeared nor­mal after 12 subcultures. On the other hand, shoots of the GA-13 rootstock produced a high incidence of vitrified and aberrant shoots after the third sub­culture and consequently multiple re­cultures at one-week intervals were necessary to maintain a stock of healthy shoots. Improvement in shoot quality following multiple reculturing at short intervals, is also reported for Juglans (Driver & Kuniyuki, 1984) and Sequoia (Fouret et al, 1984). In this investigation, vitrification was observed in the avocado shoots, probably due to a high level of endogenous BA, as it has been reported in Prunus by Alderson et al (1985).

 

Standardi (1982) working with Acti­nidia and Sriskandarajan et al (1982), with Malus, reports an increase in rotting capacities with increase in the number of subcultures. No such in­crease was observed in the two root­stocks studied in the present work, even though for the GA-13 rootstock, shoots with 29 subcultures were tested. The different rooting percentages of the IV-8 and the GA-13 rootstocks, 30 per cent and 5 per cent respectively, could be genetical in origin. However, it could also be the result of the shoots of the locally selected IV-8 rootstock coming from the tree base and the fact that this type of shoot generally remains in the juvenile state (Gillespie, 1956).

 

The grafting of adult buds onto juvenile stocks is a technique used to induce rejuvenation in grafted scions (Franclet, 1979; Pliego-Alfaro, 1981). Work is in progress at present to deter­mine the rooting threshold that might be obtained through successive grafting of GA-13 buds onto seedlings germinated in vitro.

 

REFERENCES

1      Alderson, PG, Harbour, MA & Patience, PA, 1985. Micropropagation of Prunus tenella 'Firehill'. Symposium on: in vitro Problems Related to Mass Propagation of Horticultural Plants. Gembloux. September. Abstract 15.

 

2      Anderson, WC, 1984. A revised tissue culture medium for shoot multiplication of rhododen­dron. Journal of the American Society for Horticultural Science, 9(3), 343-347.

 

3      Ben-Ya'acov, A, 1976. Avocado rootstock­-scion relationship: a long-term, large-scale field research project. V. Final report on some orchards planted during the years 1960-­1964. Calif Avocado Soc Yrb, 122-133.

 

4      Driver, JA & Kuniyuki, AH, 1984. In vitro propagation of Paradox walnut rootstock. HortScience, 19(4), 507-509.

 

5      Fouret, Y, Arnaud, & Larrieu, C, 1984. Rajeunissement in vitro du Sequoia semper­-virens. Annales AFOCEL, 111-137.

 

6      Franclet, A, 1979. Rajeunissement des ar­bres adultes en vue de leur propagation vegetative. Eludes et Recherches, 12, 3-18.

 

7      Gamborg, OL, Miller, RA & Ojima, K, 1968. Nutrient requirements of suspension cultures of soybean roots cells. Experimental Cell Research, 50, 151-158.

 

8      Gillespie, HL, 1952. A-sexual reproduction of rootstocks from heavy producing Fuerte avocados. Calif Avocado Soc Yrb, 97-101.

 

9      Gillespie, HL, 1956. Preliminary investigation of 'Residual Juvenility' in avocado seedling stems. Calif Avocado Soc Yrb, 40, 132-134.

 

10    Hartmann, HT & Kester, DE, 1983. Plant Propagation: Principles and Practices. Pren­tice-Hall.

 

11    Kadman, A, 1968. Selection of avocado rootstock suitable for use with saline irrigation water. Calif Avocado Soc Yrb, 145­-147.

 

12    Kadman, A & Ben-Ya'acov, A, 1980. GA-13 avocado rootstock selection. HortScience, 15(2), 206.

 

13    Margara, J, 1984. Bases de la multiplication vegetative. INRA. Versailles. Paris.

 

14    Molnar, GY, 1985. A new method for mass propagation of shoot cultures. Symposium on: in vitro Problems Related to Mass Propagation of Horticultural Plants. Gem­bloux. September. Abstract,  21.

 

15    Monselise, SP & Goldschmidt, EE, 1982. Alternate bearing in fruit trees. In: Horti­cultural Reviews, 4, 129-173, Ed. J Janick.

 

16    Murashige, T, 1978. The impact of plant tissue culture on agriculture. In: Frontiers of Plant Tissue Culture, 15-27. Ed. TA Thorpe, University of Calgary.

 

17    Murashige, T & Skoog, F, 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia Plan­tarum 15 473-497

 

18    Pliego-Alfaro, F, 1981. A morphogenetic study of the avocado (Persea americana Mill) in vitro. PhD Thesis, University of California, Riverside.

 

19    Sriskandarajan, S, Mullins, MG & Nair, Y, 1982. Induction of adventitious rooting in vitro in difficult-to-propagate cultivars of apple. Plant Science Letters, 24, 1-9.

 

20    Standards, A, 1982. Effetti di subcolture ripetute in germogll dl Actinidia chinensis (PI) coltivati in vitro. Rivista delta Ortofloro­frutticoltura Italiana, 66(6), 419-429.

 

21    Viseur, J, 1985. Micropropagation of pear, Pyrus communis L. in a double-phase medium. Symposium on: in vitro Problems Related to Mass Propagation of Horticultural Plants. Gembloux. September. Abstrat, 20.