1.3 Crabapple (Malus spp.)

Transcription

1 1.3 Crabapple (Malus spp.) S. SINGHAl 1 Introduction Crabapples are a member of the family Rosaceae and their primary importance is as ornamental trees because of their attractive flowers and fruits (den Boer 1959). Crabapples also have value as potential pollinizers in commercial apple orchards (Crassweller et al. 1980) and as indicator plants for the detection of latent apple viruses (Gilmer et al. 1971). Crabapples can be used as rootstocks for apples; for example, Malus robusta 5, which has the desirable feature of withstanding low temperatures would be suitable in areas where winter hardiness is a problem (Tukey 1964). The genus Malus contains about 25 species (Bailey and Bailey 1976) and includes both apples (M domestica) and crabapples. The nomenclature of crabappies is confusing because they include not only many species but also a vast array of hybrids between species and this can result in plants being mislabelled. In an attempt to rectify this problem, Jefferson (1970) reported on the history and progeny of documented crabapple species and cultivars and provided brief descriptions of them. Crab apples are subject to the same diseases as apples. These include fireblight, caused by the bacterium Erwinia amylovora, and apple scab caused by the fungus Venturia inaequalis. Fireblight can be an extremely devastating disease resulting in the loss of a major portion of the tree or even the entire tree. Apple scab, although not as debilitating, can result in unsightly lesions on leaves and disfigured fruit. It can be controlled by timely applications of fungicides. Although chemical control for these and other diseases is available, a more desirable alternative is the utilization of crabapple cultivars resistant to these problems. Nichols (1986) has conducted an ongoing survey for over 20 years of crabapples resistant to fireblight, scab, cedar-apple rust and powdery mildew. Based on his evaluations of disease resistance and aesthetic rating, outstanding performers include M sargentii, M sieboldii var. zumi cv. Calocarpa and the hybrids Liset, White Angel and Mary Potter. 1 Division of Plant and Soil Sciences, 1090 Ag Sci, P. O. Box 6108, West Virginia University, Morgantown, WV 26506, USA Biotechnology in Agriculture and Forestry, Vol. 5 Trees II (ed. by Y. P. S. Bajaj) Springer-Verlag Berlin Heidelberg 1989

2 Crabapple (Malus spp.) Crabapple Propagation and Improvement: Conventional Versus Nonconventional Methods Most crabapples propagated from seed will be dissimilar to their parents. Clonal propagation is accomplished by budding or grafting the desired scion cultivar onto seedling or c10nally multiplied rootstocks of the genus Malus. Rootstock cultivars like Robusta 5 can be c10nally propagated by layering in stoolbeds. Some crabapples, including M. toringoides and M. hupehensis, are apomictic and can be c10nally perpetuated through seed. Thchniques of shoot tip culture have been successfully developed for the propagation of crabapples and provide a means of rapid propagation of self-rooted trees. Recent reports on anther culture, isolation of protoplasts and the regeneration of plants from callus in many tree species (see Bajaj 1986), indicate that these techniques can be adapted for crabapple propagation and improvement. With further research these methods should provide a means for exploiting somaclonal variations, somatic hybridization and haploid plant production as a tool for breeding and improvement of crabapples. 2 In Vitro Culture of Crabapple Since the initial report on embryo culture of crab apples (Nickell 1951), other approaches including shoot-tip culture, anther culture and protoplast culture have been investigated. From a practical stand point the technique of shoot-tip culture has been successfully developed for rapid plant propagation. Other in vitro approaches like anther and protoplast culture need further research before their potentials can be fully exploited. These studies have been summarized in Thble Shoot Tip Culture A number of crabapple cultivars have been micropropagated through shoot-tip culture (Norton and Boe 1982; Singha 1982a; Wanstreet 1982). The procedures for explant sterilization and culture initiation utilized in these studies were fairly similar. None of these investigations was conducted with the objective of virus elimination and the shoot tip explants exceeded 1 cm in length. The surface sterilization procedures involved a min immersion in 100,10 Clorox (0.50,10 sodium hypochlorite) followed by two to three rinses with sterile water. Norton and Boe (1982) used Linsmaier and Skoog (1965) medium and obtained a maximum proliferation rate of 4.5 shoots per explant in 4 weeks with Dainty crabapple on medium supplemented with 1 mg/l benzylamino purine (BA) and similar results with 2.5 mg/l BA in Golden Hornet. Rooting of in vitro-produced shoots was obtained on basal medium containing 5 or 10 mg/l indolebutyric acid (IBA) and the rate of root initiation was enhanced by incubating cultures in the dark for 1 week prior to placing them under illumination. Rapid shoot proliferation of the cultivars Almey, Eleyi, Hopa and M sieboldii var. zumi cv. Calocarpa was

3 32 S. Singha Table 1. Summary of in vitro studies with crabapple Type of culture Basal Objective of study Reference medium" Shoot tip culture LS Micropropagation of Dainty and Golden Norton and Boe (1982) Hornet MS Micropropagation of Almey, Eleyi, Hopa Singha (1982a) and M. sieboldii var. zumi Calocarpa MS Comparative shoot proliferation of 11 Wanstreet (1982) cultivars. Micropropagation of David and Snowdrift MS Influence of agar concentration on shoot Singha (1982 b) proliferation MS Influence of agar brands on shoot pro- Singha (1984) liferation MS Mineral nutrition of shoots cultured on Singha et al. (1985) different agar medium MS Screening cultivars for frreblight resistance Wanstreet (1982) Embryo culture NI Rapid development of seedlings for evalua- Nickell (1951) tion Anther culture MS and Regenerating haploid plants Wu (1981) WH Callus culture AS Callus fusion between different species Fujii and Nito 1972) MS Sorbitol as a carbon source for callus Chong and Taper growth (1972) MS Influence of 13 carbohydrates on callus Chong and Taper growth (1974a) MS Influence of light intensity on sorbitol Chong and Taper metabolism and callus growth (1974b) MS Comparative growth of callus of Rosaceae Coffin et al. (1976) species on sucrose and sorbitol MS Organogenesis in callused leaf explants Bates (1986) Protoplast Protoplast isolation from cotyledons, sus- Bates (1986) pension and shoot cultures Basal Media Abbreviations. AS = Central Agric. Expt. Stn. (refer to Fujii and Nito (1972); LS = Linsmaier and Skoog (1965); MS = Murashige and Skoog (1962); NI = Nickell (1951); WH = White (1943). a This refers only to basal nutrient media. Organic and hormonal supplements vary depending on response elicited during the investigation. achieved by Singha (1982a) on MS salt mixture (Murashige and Skoog 1962) supplemented with 1 or 2 mg/l BA (Fig. 1). Higher BA levels (4 or 8 mg/l) resulted in good shoot proliferation but a greater percentage of the shoots were small or rosetted (Thble 2). Excellent rooting of in vitro-derived shoots was obtained on half-strength MS medium containing 0.1 or 0.2 mg/l naphthaleneacetic acid (NAA) (Fig. 2a, Thble 3). Higher NAA concentrations resulted in an inhibition of root elongation and a lowered quality of roots. Plants of all four cultivars were successfully acclimated to ambient conditions (Fig. 2 b). Wanstreet (1982) compared the shoot proliferation response of 11 crabapple cultivars on MS medium containing 2.5 mg/l BA and 0.1 mg/l NAA. Whereas the cultivar David produced 15 shoots per explant over an 8-week period, no multi-

4 Crabapple (Malus spp.) 33 Fig. 1. Shoot proliferation of crabapple after 8 weeks of MS medium containing 2 mg/i BA Table 2. The effect of BA concentrations on shoot proliferation of four crabapple cultivars after 8 weeks on MS medium. (Singha 1982a) BA (mg/i) Shoots per tip Almey Eleyi Hopa Calocarpa Total >0.5cm Total >0.5cm Total >0.5cm Total >0.5cm plication was obtained in either Adam or M. sargentii (Fig. 3). Shoots of the cultivars David and Snowdrift were rooted on MS medium containing 1 mg/l NAA or by planting shoots in a peat-perlite mixture with or without a 15-s treatment in 100 mg/l IBA. The influence of the physical state of the culture medium on shoot proliferation of Almey crabapple was studied by Singha (1982b, 1984). In initial investiga-

5 34 S. Singha a Fig. 2. a Five-week-old crabapple shoot rooted on half-strength MS medium containing 0.1 mg!l NAA. b Crabapple plant regenerated from shoot tip culture b Table 3. The effect of NAA concentrations on rooting of four crabapple cultivars after 4 weeks on half-strength MS medium. (Singha 1982a) NAA Rooting (070) (mg!l) Almey Eleyi Hopa Calocarpa

6 Crabapple (Malus spp.) 35 Fig. 3. Comparative shoot proliferation of II crabapple cultivars after 8 weeks on MS medium containing 2.5 mg! 1 BA and 0.1 mg! 1 NAA. (Wanstreet 1982) M. 'Adam.' M. 'Arrow ' M. 'Centennial' M. 'David' M. 'Indlan Magic' M. 'Lemoine'!!. sargentll M. 'Mary POlTer' M. 'Snowdrift' M. 'Von Eseilin. M. 'Winter Gold ' TOTAL NUMBER OF SHOOTS PER INITIAL Fig. 4. Influence of Phytagar concentration on shoot proliferation of AImey crabapple after 8 weeks on MS medium containing 2 m g!1 BA. Agar concentration (left to right) 0, 0.3, 0.6, 0.9 and (Singha 1982b) tions shoots produced in vitro were cultured on MS medium containing 2 mg/ l BA and Phytagar (Grand Island Biological Company, Grand Island, NY 14072) levels ranging from 0 to 1.211,10. The greatest shoot proliferation and growth occurred on medium containing 0.3% agar (Fig. 4) and increasing agar concentrations decreased both shoot proliferation and shoot growth (Singha 1982 b). Similar results were obtained when TC agar (KC Biological, Lenexa, KS 66215) and Bacto-agar (Difco Laboratories, Detroit, MI 48232) were compared over the same concentration range (Singha 1984). However, the reduction in shoot proliferation and growth at higher agar concentrations was especially severe with Bacto-agar (Fig. 5). Singha et al. (1985) determined the mineral nutrient composition of shoots proliferated on MS medium containing varying concentrations of these three brands of agar. Large variations were observed in many elements both in these agar brands, and in explants cultured on media containing similar concentrations of different agar brands (Fig. 6). The variations in shoot proliferation and explant growth, however, could not be explained on the basis of variations in individual elements. They hypothesized that from a nutritional standpoint, the

7 36 S. Singha 20 j 15 VI 0 10 d Z : ~ 1.4.c ~ T! TC AGAR ~. BACTO-AGAR I\I_~,/'t-, '! (~ \ ""I \ ~f ~--_I --I ~280!240 1:200 f >- Q I /\'~,!~ \! 1---_ Agar Concentration (percent) Fig. 5. The effect of TC agar and Bacto-agar on shoot proliferation, fresh weight, and dry weight of Malus Almey; after 8 weeks on MS medium containing 2 mg/i BA. Vertical bars represent SE. (Singha 1984) alteration of the nutrient composition of the basal medium by the addition of different agars best explained the variations induced by them. These and other studies (Debergh et al. 1981; Debergh 1983) clearly demonstrate that both brand and concentration of the agar exert a strong influence on shoot proliferation and should be considered from a standpoint of more than simply a means of solidifying the culture medium. An interesting application of shoot tip cultures was their utilization to screen Malus species for fireblight resistance (Wanstreet 1982). Shoot tips of Arrow, David and Winter Gold crabapple and Jonathan apple were inoculated with suspensions of Erwinia amylovora. Jonathan, which has a high susceptibility to fireblight in the field, demonstrated similar susceptibility in vitro, whereas David, which has a higher resistance in the field, showed higher resistance in culture. Although the field resistance of Arrow did not correlate with results obtained in vitro, this technique has the potential for being adapted as a screening method for fireblight resistance under defined environmental conditions.

8 Crabapple (Malus spp.) 37 4.lIO 4.00 i 3.lIO ~ " 3.00 ~ :l<: 2.lIO 2.00 * _ V= :52X+O,0488X V= X-4-0,0427X A V = ISIX X2,... '"...,... \',... ~, ",..., _----,,' l.lio L-L- ---I....L... "- ---I...J l1 i ~ 0.40 " i O.3~ l ~ 0.50 o :i.! u " * ,,,, /'. ~~ I " / / " a //.. I,/ I, ,,' ",," I "".,',...~ yco.o4+0,08ix-o.004gx t' Y::IIO.06 +O.080)(-O.0037X~... Y=O.Z X-O.0004X 1.2, y=o.7s X+O.0048)(2 "---e V= )( X2 A- --A 1= X+O.OO4GX2 "- '"" "- ~,..'......~----~... -~-----, o Fig. 6. Macronutrient concentration on a dry weight basis in Malus Almey shoots proliferated on MS medium containing Bacto-agar (_---_), Phytagar ( ) and 1C agar (.A A.), and comparative values in liquid medium (*). Second-order equations are shown regardless of level of significance. (Singha et al. 1985) ~ 0.19 > i 0.17 '" 0.16 :::i: O.lll 0.14 y= X )( Y= X )(2 " ~... YI:O )( X 2,,"' * t:: L- "- -L-..-.JL..::J o Aoar Conc@ntration ("10)

9 38 S. Singha 2.2 Embryo Culture As with many other fruit trees in the family Rosaceae, the earliest tissue culture studies with crabapple were conducted using embryos. Nickell (1951) successfully cultured embryos excised from mature fruit of a crabapple tree with a weeping growth habit. The objective of the study was to reduce the time for establishing seedlings which could be evaluated for their growth habit. Seeds were obtained from fruit, which had been surface sterilized with ethanol. After removing the seed coat, the embryos were cultured on a hormone-free nutrient medium containing 2% sucrose and 1 % agar. The embryos grew rapidly and seedlings were transferred to soil in 4 weeks. Whereas seeds planted directly in the soil were just beginning to germinate after 9 months, those produced through embryo culture were over 1 m in height. The removal of the seed coat was necessary to induce rapid growth and seeds with intact seed coats failed to germinate even after 1 year in culture. The removal of a portion or the entire cotyledon, however, did not adversely influence germination or growth of embryos. 2.3 Anther Culture Anthers of Huang Thi-ping and Jin Hong crabapple containing microspores in the late-uninucleate stage were cultured by Wu (1981) on MS medium containing either 1-2 mg/l IAA, NAA or 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/l kin. Following callus formation, the cultures were transferred to White's medium containing 1-2 mg/l BA and mg/l NAA. Shoots regenerated from callus of Huang Thi-ping were rooted and 28 plantlets acclimated to ambient conditions. Based on chromosome counts, three of seven regenerated plants examined were confirmed to be haploid. 2.4 Callus Culture Fujii and Nito (1972) investigated the fusion of calli from different plant species to determine their graft compatibility. Internodal sections were sterilized by immersion in 70% ethanol for 2-3 min, 0.8% sodium hypochlorite for 4 min and rinsed with sterile water. After removal of the bark, explants were cultured on medium containing 1 mg/l NAA and 1 mg/l kin for 2-3 weeks. Calli were subcultured on medium supplemented with 2 mg/l NAA and 10% coconut milk and placed in contact with calli of other species. Excellent callus fusion was obtained between apple cultivars and M prunifolia. However, good fusion also occurred between Pyrus serotina and M prunifolia. They concluded that the fusion of calli of different species often failed to reflect the botanical variation between these species. Chong and Taper (1972, 1974a) investigated the effectiveness of sorbitol and other carbohydrates on callus growth of the apple cultivars Cortland and McIntosh and the crabapple rootstock Robusta 5. Internodal explants were sterilized by immersion in 95% ethanol and after removal of the bark were cultured on MS

10 Crabapple (Malus spp.) 39 medium containing 2 mg/l NAA and 0.2 mg/l kin. The growth of Robusta 5 on either 30/0 glucose or sorbitol was superior to that on sucrose, demonstrating that sorbitol was an excellent carbon source (Chong and Taper 1972). When 13 different carbon sources were compared, 6% fructose induced the best callus growth in Robusta 5 and 6% glucose or sorbitol were superior to sucrose (Chong and Taper 1974a). However, with Geneva crabapple, both callus initiation and growth were similar on media supplemented with either 3 % sucrose or sorbitol. While sorbitol can be an effective substitute for sucrose for both callus initiation and growth in Malus species, this does not extend to most other species of Rosaceae (Coffin et al. 1976). The growth of Robusta 5 callus and the pattern of accumulation of various carbohydrates was not influenced by light intensity over a range of 0 to 7800 Ix (Chong and Thper 1974b). Leaf explants obtained from seedlings of M hupehensis were surface sterilized with 10% Clorox for 10 min, rinsed three times with sterile water and cultured on MS medium containing 1 mg/l NAA and 1 mg/l kin (Bates 1986). After 2 weeks, callused leaf sections were transferred to medium containing NAA (0.5 to 16 mg/l) and placed either in the darkness or under 16 h illumination. Root initiation was observed at all NAA concentrations in the dark and was superior to rooting in cultures maintained under lights. Shoot initiation was obtained in 1 of 5 calli which had been grown on medium containing 11 mg/l 2,4-D, 0.5 mg/l kin and 300 mg/l glutamine for 5 days and then placed on 2 mg/l BA. The shoot was sub-cultured, proliferated and rooted, and plants acclimated to ambient conditions. Various other shoot initiation treatments were unsuccessful. 2.5 Protoplast Culture Bates (1986) isolated protoplasts of M hupehensis. The optimum protoplast yields and viability being obtained when cotyledons were incubated for 3 h in an enzyme mixture containing 3% cellulysin, 1 % macerase and 0.6M sorbitol. When different source tissues were compared, cotyledons gave high yields of viable protoplasts, in vitro-derived shoots gave lower yields and cell suspensions were a poor source of protoplasts. Although various plating techniques were utilized, callus induction was not obtained. 3 Conclusions and Prospects Micropropagation of many crabapple cultivars has been achieved through shoot tip culture and this technique enables rapid production of large numbers of plants. Culturing actively growing shoot tips of most crabapple cultivars on MS medium containing 1 or 2 mg/l BA, under 16-h illumination at 25 C results in good shoot proliferation. PlantIets.can be regenerated by rooting in vitro-produced shoots on medium containing either NAA or IBA. However, further investigations are still needed with certain desirable but recalcitrant clones like M sargentii.

11 40 S. Singha Although protoplasts have been isolated, no further development has been obtained. Some success has been achieved in callus organogenesis, but the predictability of plant regeneration needs to be greatly enhanced. This remains a necessary prerequisite before these approaches can be used for the improvement of crabapple. The successful regeneration of haploid crabapple plants through anther culture should be of value in non-conventional plant breeding. While many obstacles yet need to be overcome, these studies indicate that both anther culture and protoplast culture can provide powerful tools for genetic manipulation and improvement of crab apples. References Bailey LH, Bailey EZ (1976) Hortus third: a concise dictionary of plants cultivated in the United States and Canada. MacMillan, New York Bates RM (1986) Callus organogenesis and protoplast isolation in Malus hupehensis. MS Thesis, West Virginia Univ, Morgantown Bajaj YPS (ed) (1986) Biotechnology of tree improvement for rapid propagation and biomass energy production. In: Biotechnology in agriculture and forestry, vol 1. 1tees I. Springer, Berlin New York Thkyo Chong C, 1liper CD (1972) Malus tissue cultures. I. Sorbitol (D-glucitol) as a carbon source for callus initiation and growth. Can J Bot 50: Chong C, 1liper CD (1974a) Malus tissue cultures. II. Sorbitol metabolism and carbon nutrition. Can J Bot 52: Chong C, 1liper CD (1974b) Influence of light intensity on sorbitol metabolism, growth and chlorophyll content of Malus tissue cultures. Ann Bot (London) 38: Coffin R, 1liper CD, Chong C (1976) Sorbitol and sucrose as carbon source for callus culture of some species of the Rosaceae. Can J Bot 54: Crassweller RM, Ferree DC, Nichols LP (1980) Flowering crab apples as potential pollinizers for commercial apple cultivars. J Am Soc Hortie Sci 105: Debergh PC (1983) Effects of agar brand and concentration on the tissue culture medium. Physiol Plant 59: Debergh PC, Harbaoui Y, Lemeur R (1981) Mass propagation of globe artichoke (Cynara scolymus): Evaluation of different hypotheses to overcome vitrification with special reference to water potential. Physiol Plant 53: den Boer AF (1959) Ornamental crab apples. Am Assoc Nurserymen, Washington, DC Fujii T, Nita N (1972) Studies on the compatibility of grafting of fruit trees. I. Callus fusion between rootstock and scion. J Jpn Soc Hortic Sci 41:1-10 Gilmer R, Mink 01, Shay JR, Stouffer RF, McCrum RC (1971) Latent viruses of apple. I. Detection with woody indicators. Search (Agric) 1(10):1-21. NY State Agric Exp St, Geneva, NY Jefferson RM (1970) History, progeny and location of crabapples of documented authentic origin. Nat Arboretum Contrib 2. US Dep Agric, Washington, DC Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: Nichols LP (1986) Disease-resistant crabapples. Plant pathology Contrib Penn State Univ, Univ Park Nickell LG (1951) Embryo culture of weeping crabapple. Proc AmSoc Hortic Sci 57: Norton ME, Boe AA (1982) In vitro propagation of ornamental rosaceous plants. HortSci 17: Singha S (1982a) In vitro propagation of crabapple cultivars. HortSci 17:

12 Crabapple (Malus spp.) 41 Singha S (1982b) Influence of agar concentration on in vitro shoot proliferation of Malus sp. Almey and Pyrus communis Seckel. J Am Soc Hortic Sci 107: Singha S (1984) Influence of two commercial agars on in vitro shoot proliferation of Almey crabapple and Seckel pear. HortSci 19: Singha S, Townsend ED, Oberly GH (1985) Mineral nutrient status of crabapple and pear shoots cultured in vitro on varying concentrations of three commercial agars. J Am Soc Hortic Sci 110: Thkey HB (1964) Dwarfed fruit trees. Cornell Univ Press, Ithaca Wanstreet A (1982) In vitro inoculation of tissue culture propagated Malus shoot tips with Erwinia amylovora. MS Thesis, Ohio State Univ, Columbus White PR (1943) A handbook of plant tissue culture. Cattel, Lancaster, PA Wu JY (1981) Obtaining haploid plantlets of crab apple from anther culture in vitro. Acta Hortic Sin 8(4):36 (in Chinese)

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