From different combinations of basal medium and growth regulators tested, itwasfound that the best niediumfor producing shoots front mature embryos wasAI."'I 2 ppnt BAP + 2 ppnt NAil -1 I. 25 pplll IIIlI; front apical tneristems' CD +I. 12 pplll !:fA P + 0.0./ pptn 2, .J-D r- 0.01 pplll J.:; andfrom needle fascicles, cowith no growth regulator. Of 3 substrates tested for growing seedlings toproduce explants, the best (jiJl' apical-meristem explants) was low-nutrientcompost, and the best age o/' seedling was 2 I days. Explants from one-year oldplants and shoots produced in vitro gave more shoots than did those frotnmature trees; there was no significant difference in the length of the shoots.Production of adventitious budsfor rapid multiplication was greater on exp Iants0/ apical meristem origin than on those front mature embryos or needle[ascicles; the percentage survival decreased significantly in second and thirdgenerations ofexplants oral! three origins

Mayhead, G.J.

Senerathl, W.T.P.S.K.

Shaw, D.S.

University of Sri Jayewardenepura: Journals & Proceedings

Annual Forestry Symposium, 1996SHOOT INITIA nON AND MULTIPLICATIONOF CORSICAN PINE IN VITROW.T.P.S.K. Senerathl, G.J. Mayhead2, and D.S. Shaw2'Department of Botany, University of Sri Jayewardcnepura;2University of Wales, Bangor(Based on a I'hLJ thesis hy IV.7:1'..\'.A:.. Senerath submitted to the University of Wales)AbstractFrom different combinations of basal medium and growth regulators tested, itwasfound that the best niediumfor producing shoots front mature embryos wasAI."'I 2 ppnt BAP + 2 ppnt NAil -1 I. 25 pplll IIIlI; front apical tneristems' CD +I. 12 pplll !:fAP + 0.0./ pptn 2, .J-D r- 0.01 pplll J.:; andfrom needle fascicles, cowith no growth regulator. Of 3 substrates tested for growing seedlings toproduce explants, the best (jiJl' apical-meristem explants) was low-nutrientcompost, and the best age o/' seedling was 2 I days. Explants from one-year oldplants and shoots produced in vitro gave more shoots than did those frotnmature trees; there was no significant difference in the length of the shoots.Production of adventitious budsfor rapid multiplication was greater on exp Iants0/ apical meristem origin than on those front mature embryos or needle[ascicles; the percentage survival decreased significantly in second and thirdgenerations ofexplants oral! three origins.IntroductionRapid multiplication is of interest not only for clonal propagation on a large commercialscale, but also as a potential means of gene-pool preservation (Jelaska, 19S7). Aproblem in pines and other slow growing species is the long time it takes to produceusable plants by traditional vegetative propagation, due to the low number of rootablecuttings obtained from each small plant per year. For many of the species clonalpropagation by rooting cuttings is difficult, especially when the cuttings are taken fromadult trees. Corsican pine (Pinus nigra var. maritima (Ait.) Melville) is an importantplantation species in Europe. The objective of the investigation reported here was todevelop tissue-culture techniques for the rapid multiplication of this species, which hasa very slow growth rate and also a low rooting percentage. The techniques can also beused for the rapid propagation, or clonal propagation, of important forest trees in SriLanka.Materials and methodsAs the starting material for shoot initiation, mature embryos. apical mcristcms of youngseedlings, and needle fascicles were used. Thcy were washed in W'},;, hypochloritesolution, and thcn trimmed to get 2-5 nun explants. The prepared cxplants were dippedin 70% ethanol for 30 s and treated with 100 ppm HgCI2 for 3 minutes. After they hadSenarathbeen rinsed three times in distilled water, they were blotted dry on sterile filter paperbefore being transferred onto the media. For embryo culture the seeds were washed, theseed coat was removed under sterile conditions. and the embryos were excised. All thecultures were incubated at 26ve, with a lo-hour day. A completely randomized designwas used in all experiments. and the culture vessels were re-randomized at seven-dayintervals.Experiment 1 - The best medium for shoot induction from different explantsourcesThe explanrs were cultured in different basal media. ill the presence of different grov vthregulators. Different basal media and growth regulators used in experiments are given intable I. After five weeks a count was made of the percentage of explants producingshoots in each of the different media.Experiment 2 - The best substrate for growing seedlings to provide cxplantsSeedlings were raised for 21 days in three different substrates, to provide apices for useas cxplants. The substrates were moist paper, low-nutrient compost. and sterile-wateragar. After five weeks of incubation. a count was made of the number of explants fromeach source that produced shoots, and the shoot lengths were measured.Experiment 3 - The best seedling age to collect explantsThe best substrate for raising seedlings were determined from experiment 2. Apiceswere collected from seedlings grown in this substrate to different ages: 7, 14.21. and 28days. After 5 weeks of incubation a count was made of the number of explantsproducing shoots. and the shoot lengths were measured.Experiment 4 - Shoot initiation from needle fasciclesNeedle fascicles from mature trees. from one-year-old plants. and from shoots producedin vitro were cultured in media.in order to determine the rate of shoot initiation. Afterfive weeks a count was made of the number of cxplants producing shoots. and the shootlengths were measured.Experiment 5 - Shoot multiplicationForty shoots of each of three explant origins (mature embryos. apical meristems andneedle fascicles) were cultured in a bud-induction medium to initiate adventitious buds.After six weeks a count was made of the number of adventitious buds per explant. andten from each explant were sub-cultured as the second generation. The same procedurewas followed for a third generation. In each generation a count was made of thepercentage survival, and of the number of adventitious buds per shoot.Annual Forestry Symposium, 1996Table l: Different basal madia :\I1d growth regulators usedBasal media Growth regulatorsMurashige and Skoog (MS) IBACampbell and Oyrzan (CD) BAP. NAAJuglans Medium (OKW) 2.4 -0Mc Cown Woody Plant Medium (WPM) GA), KResultsExperiment IOn the different combinations of basal media and growth regulators that were tested.mature embryos showed the best shoot-initiation performance in MS + 2.0 ppm BAP +2.0 ppm NAA + 1.25 ppm IBA. The best medium for shoot initiation from apicalmcristcms was CD + I. 12 ppm BAP + (l.04 ppm 2,4-0 + (H) I ppm K. For needlefascicles the best was CD with no growth regulator (Table 2).Table 2 : Shoot initiation in vitro from different explant sources in different culturemediaMedium Percentage of explants producing shootsMature crnhrvns Apical meristems Needle fasciclesI 50* 100 602 75* 70 703 45* 90 SO"callus productionMediaI = CD + 1.12 ppm BAP + 0.04 ppm 2,4-0 + 0.01 ppm K2 = MS + 2.0 ppm BAP + 2.0 ppm NAA + 1.25 ppm IBA3 = CD. no growth regulatorExperiment 2The results of this experiment showed that there was a significant difference betweenthe three substrates in the number of cxplants producing shoots. Although all thecxplants (20 out or 20 ) of apical meristems collected from seedlings raised on compostand water agar produced shoots. the apices collected from seedlings raised on compostgave statistically significantly greater shoot extension when compared with the othertwo substrates. As Table 3 shows. therefore, low-nutrient compost is the best of thesethree substrates for raising seedlings to provide apices for shoot initiation.SenarathT ••ble J : Effect of different seedlin~ substrates on shoot initiation from apicalmcristemsSeedling substrate Numher of cxplants producim;shootsMcan shoot lenJ..rth(mm)Moist paperCompostWater agar1221l21lI·U22.57.1Expcrimcut JThere were significant differences in the number of cxplauts producing shoots and inthe mean shoot length according to the age of the seedlings. Very young seedlings (7days) produced only callus: there was no shoot elongation. The mean shoot lengthincreased up to 21-day seedling age, and declined at 2X days (Table 4).Tahle": Effect of secdling lIge on shoot initilltion in vitro from apical meristemsA~e of seedlin~ Number of explants Mean shoot leng••hproducing shoots (mm)7142128callus only1720147.221.815.4Experiment .•The age of the source of the explant had a significant effect on shoot uuuatron fromneedle fascicles. Between the three different origins there was a significant dilTerence inthe number of cxplanis producing shoots. but 1I0t in the mean shoot length. Needlefascicles produced in vitro had a greater tendency to initiate shoots than those fromplants which were more than one-year-old (Table 5).Table S .Shuot initiation frnm needle fasciclesSource of explant Numher of cxplantsproducing shootsMean shoot lenJ..rth(mm)Mature treesOne-year-old plantsShoots produced ill vitro820205.205.656.24Experiment SFor all three origins (mature embryo. apical mcristcm and needle fascicle) an apparentreduction in the mean number of shoots per generation was not significant. The overallmean for the number of buds per shoot was significantly greater [or shoots of apicalmcristem origin. The percentage survival decreased significantly for all three originsfrom the first to the third generation (Table (,).Annual Forestry Symposium, 1996Table 6 : Mean number of adventitious buds per shoot (and percentage survival)after 18 weeks. i.e. three generations of six weeks eachGeneration Mature embryo Apical meristem Needle fascicle21.20 (100%)I.n (876%)0.90 (75.5'}';,)2.75 (100%)1.73 (89.8%)1.40 (80.6%)1.55 (100%)1.46 (86.4'1'0)1.20 (0.11%)3DiscussionSeedling substrateThe main problem in using non-sterile substrates was contamination of the cultures.Subjectively. there was also a visual difference between seedlings grown on differentsubstrates. Seedlings grown on compost were dark green in colour and more robust.perhaps because of nutrients present in it. Seedlings [rom moist paper. which had nonutrients added. were more tender. The seedlings in water agar were of a lighter greenand weaker in appearance. Tranvan (1979) has reported a correlation between thegrowth of the parent plants and callus production from the explants.Seedling ageThe length of the shoots produced [rom mcristcm cxplants increased up to a seedlingage of 21 days. and then declined. In contrast to the above results. Zel et al. (1988)found that explants [rom seedlings of different ages (three. six. and nine weeks) did notdisplay a wide range of variation in their shoot elongation, but did vary more inadventitious bud formation.Needle fascicle cultureNeedle fascicles from younger plants had a greater tendency to initiate shoots than thosefrom mature plants. Jasson & Borman (1981) similarly found that bud induction inneedle explants of Picea abies was greatly affected by needle age. Von Arnold &Eriksson (1979) obtained good results with Picco abies needle explants collected justafter the bud flush.Shoot multiplicationTranvan (1979) attempted to induce adventitious bud formation at the apex of veryyoung Pinus sylvestris seedlings. Although multiple buds were formed. it was not clearwhether bud induction was occurring directly at the seedling apex, on the cotyledons. oron internodal tissue. Webb (1978) found that in Pinus contorta apices several of theadventitious shoots started to grow but only one or two continued to extend. No recordsSenarathwere found in the literature on the decline of survival ability from one generation to theother, but it can be explained by the decreasing totipotency of cells with time.ReferencesArnold, S. van and Eriksson, T. (1979). Bud induction on isolated needles of Piceaabies grown in vitro. Plant Science Letters 15, 363-372 .Jasson, E. and Bornman, C.H. (1981). In vitro initiation of adventitious structures inrelation 10 the abscission zone in needle explants of Picea abies - anatomicalconsiderations. Physiologia Plantarum 53,191-197 .Jelaska, S. (1987). European pines. In J.M. Bonga and Durzan (Eds.), Cell and tissueculture inforestry, Netherlands, Martinus Nijhoff. Pp. 42-60.Senarath, W.T.P.S.K (1996) In vitro propagation of Pinus nigra var. manuma(Corsican pine). Ph.D Thesis, University of Wales, Bangor, UKTranvan, H. (1979). 1n vitro adventitious bud formation on isolated seedlings of Pinussylvcstris. Biologia Plantarum 21, 230-233.Webb, K..J. (1978). Growth and morphogenesis of lissue cultures o/Picea sitchensis andPinus contorta. PhD thesis, University of Leicester.ZeL L Gogala, N. and Camloh, M. (1988). Micropropagation of Pinus sylvestris. PlantCet! iissue ana Organ Culture 14. 169-175.--------

SHOOT INITIATION AND MULTIPLICATION OF CORSICAN PINE IN VITRO

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SHOOT INITIATION AND MULTIPLICATION OF CORSICAN PINE IN VITRO

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