In vitro Propagation and In vivo Acclimatization of Three Coffee Cultivars (Coffea arabica L.) From Yemen

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In vitro Propagation and In vivo Acclimatization of Three Coffee Cultivars (Coffea arabica L.) From Yemen
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  World Applied Sciences Journal 2 (2): 142-150, 2007ISSN 1818-4952© IDOSI Publications, 2007 Corresponding Author: Dr. Mohamad Shatnawi,Department of Biotechnology, Faculty of Agriculture Technology, Al-BalqaApplied University, Al -Salt 19117, Jordan 142  In vitro Propagation and  In vivo  Acclimatization of Three Coffee Cultivars( Coffea arabica L.) From Yemen  Naji Ebrahim, Rida Shibli,Ibrahim Makhadmeh, Mohamad Shatnawi and Abdallah Abu-Ein 11 12 College of Agriculture, Jordan University of Science and Technology, Irbid, Jordan 1 Department of Biotechnology, Faculty of Agriculture Technology, 2 Al-Balqa Applied University, Al-Salt 19117, Jordan Abstract:  Micropropagation of  Coffea arabica  cvs. Oudayni, Hammady and Dawaeiry from Yemen wereinitiated from seeds. Seeds were surface sterilized and inoculated into media supplemented with differentsalt strengths and germinated under dark. Seeds germinated on agar medium gave high hypocotyl length,high root length and full cotyledonary leaves expansion after 120 days of culture. Proliferation of thesecultivars was experimented on MS media supplemented with different levels (0.0, 2.0, 4.0. 6.0 or 8.0 mg l) of  1  N6-Benzyladenine (BA), Thidiazuron (TDZ), 6-furfurylaminopurine (Kinetin), 6-(4-hydroxy-3-methyl-2- butenylamino) purine (Zeatin) or 6-(,-Dimethylallylamino) purine (2ip). Highest proliferation for all cultivarswas obtained when BA was used at the highest level (8.0 mg l). Satisfactory proliferation rate in the three 1 cultivars was achieved at 8.0 mg l kinetin and 6.0 mg l TDZ. Zeatin and 2ip were both failed to promote 11  proliferation at any used level. Rooting was experimented on half-strength MS media supplementedwith different levels (0.0, 2.0, or 3.0 mg l) of indole-3- butyric acid (IBA), indole-3- acetic acid (IAA) or  1 1-naphthaleneacetic acid (NAA). Highest root number and length was achieved at 3.0 mg l IAA or IBA for  1 all cultivars. Rooted plantlets were transferred to 1 peat: 1 perlite mixture and ex vitro acclimatization gave 100%survival. Key words:  Acclimatization coffee in vitro  micropropagation root formation INTRODUCTION C. dewevrei  in Ivory Coast and Zaire. These later Coffee( Coffea arabica ) is one of the most importantacceptable only in local markets [4]. In Yemen, there areagricultural products in the international markets.many coffee cultivars and they are named according toThe world trade in coffee is an important contributor totheir place of origin, for example Oudayni, Matiery,the income of some fifty or more producing countries onHammady, Yavaeiy. In addition to the previous cultivarswidely differing scales There are many countries whichof arabica coffee which grow to a standard size, there isdepend in their economy on the coffee exportation [1]a dwarf cultivar which is known as Dawaeiry in YemenCommercially, only two out of more than 100 coffeeand universally known as Mokka [5]. species are cultivated: the C. arabica (Arabica) andBotanically, coffee belongs to the family Rubiacea C. canephora  (Robusta) [1, 2, 3]. Quality beverage isandis classified taxonomically under the genus Coffea  produced from C. arabica  which is cultivated at higherwhich includes at least 64 species grouped into four altitudes. This species represent 70% of the commercialsections [6]. The most important species is C. arabica .coffee of the world and about 99% of Latin AmericanThe coffee from Yemen gave rise to two distinct types: production.On the other hand C. canephora  is usually C. arabica  var. arabica, usually called "typical", whichgrown in tropical areas at lower altitudes whichwas the earliest grown coffee in Asia and Latin Americarepresent 80% of the African production. On a veryand C.arabica var. bourbon which came to Southreduced scale, C. liberica  is grown in some AfricanAmerica through the island of La Reunion, formerly calledcountries, C. racemosa  is grown in Mozambique andBourbon [7].species produce beans of lower quality that are  World Appl. Sci. J., 2 (2): 142-150, 2007  143Recently invitro  culture has played an important role  In vitro  germination:  For  in vitro germination of seeds,in agriculture and plant science. This method allows the production of large number of genetically identical plantswhich can be produced from a single mother stock [8].Plant production via tissue culture is advantageous over traditional propagation methods because it leads to the production of disease and virus - free plants [8, 9]. Also,it allows the production of a high number of plants, in ashort period of time and in a very limited propagationspace [9]. In addition, rapid multiplication rate of plantsthat are difficult to propagate conventionally can be easilyachieved via in vitro culture [10].Various approaches have been considered for  in vitro  multiplication of coffee ( C. arabica ) apicalmeristem and axillary bud culture, induction anddevelopment of adventitious buds [10] and somaticembryogenesis [11]. C. arabica  is predominantly self-pollinated [12] andthus, the progenies as arising from the seeds are veryuniform. Therefore, seeds are considered a goodstarting material in the in vitro  establishment for this plant. Therefore, the objective of this investigationwas to develop a protocol for in vitro  establishment,multiplication, rooting and acclimatization of someleading coffee cultivars ( C. arabica ) from Yemen. MATERIALS AND METHODSEstablishment of plant materialSeeds:  Healthy 10 years old coffee ( C. arabica ) trees inYemen were used as a source for seeds. Fruits of the threecoffee cultivars, Oudayni, Daweiry and Hammady, wereharvested in September 2001. Seeds were obtained usingmature and ripe fruits by depupling (natural fermentationfor24 hours) and washing. Seeds were then dried under shade until approximately 13% moisture content which issuitable for storage. Surface sterilization:  Seeds were surface sterilized bywashing thoroughly under running tap water for 10 min.Afterwardsseeds were immersed in Benomyl (fungicide)solution (10 mg l)for 5 min. Then rinsed with autoclaved 1 distilled water, for 5 min to remove traces of Benomyl.Seeds were immersed in the antiseptic solution of 1.25%sodium hypochlorite plus two drops of tween-20 for 20 min (under the laminar air-flow cabinet). Seeds wererinsed with sterile distilled water for three times (5 mineach). Seeds were then soaked in 70% (v/v) ethanolsolution for 30 sec and then rinsed with autoclaveddistilled water three times (5 min each).four treatments were used, Agar medium only, fullstrength medium MS [13], half strength MS mediumandQuarter strength MS medium. In the differenttreatments, media contained 8.0 g l agar (Difco-Bacto) 1 and supplemented with 2 mg l Gibberellic acid (GA). 13 Sterilized seeds of the three cultivars were inoculated in25 x 150 mm test tubes containing 25 ml of media. Eachtreatment consisted of twenty replications in a CompletelyRandomized Design (CRD). Cultures were kept under dark at 24±2 °C for 30 days until full germination. Totalgermination percentage and contamination % wererecorded. Germinated culture were transferred to thegrowth chamber under 16 hours of artificial fluorescentlight (photosynthetic photon flax density PPFD = (40-45 µmol/m/sec) and 8 hours of darkness at 26±2°C 2 for 90 days. Different parameters, including hypocotyllength, root length, total seedling length andcotyledonary leaves expansion were recorded. MicropropagationShoot multiplication:  Microshoots (10 mm) weresubcultured in Erlenmeyer flasks (250 ml) containing150 ml of solid MS media. The following experimentswere done to study the best cytokinin type andconcentration for maximizing microshoot productionfor each cultivar. Different concentrations (0.0, 2.0, 4.0,6.0 or 8.0 mg l) of BA, TDZ, Kinetin, 2ip or Zeatin 1 were studied in separate experiments. All treatmentshad 0.5 mg l IAA. The cultured microshoots were 1 incubatedunder 26±2 °C with 16 hours light(photosynthetic photon flux density; PPFD = (40-45)µmol/m / sec) and 8 hours dark. Data were collected after 120 2 dayson number of proliferated shoots, shoot height,numberof leaves/explant. Callus and the plant performance were monitored. Experiments were arrangedin a completely randomized design (CRD) with 10replications. Rooting:  Rooting was carried out for each cultivar bysubculturing microshoots (10 mm) in 25 x 150 mm testtubes containing 25 ml of solid half-strength MS mediaand sucrose (15 g l). Media was supplemented with 1 IBA, IAA or NAA at 0.0, 1.0, 2.0or 3.0 mg l. Experiments 1 were arranged in a Completely Randomized Design(CRD) with 10 replications. The cultured microshootswere maintained under 26±2 °C with 16 hours light(PPFD) = 40 - 45µmol/m/sec)and 8 hours dark. Data were 2 collected on number of roots, root length and shootheight after 60 days.  World Appl. Sci. J., 2 (2): 142-150, 2007  144 Ex vitro acclimatization:  Ex vitro  acclimatization wascarriedout by opening test tubes for 3 days beforetransferring plantlets outside of the growth chamber.  Invitro  rooted plantlets were extracted from test tubesandthe agar was removed by washing with warm sterilewater. The plantlets were transferred to 1 peat: 1 perlitemixture in 84 cell polystyrene trays covered with glass beakers for 3 weeks. Plantlets were acclimatized under 16hours supplementary light of 40-45 µmol./m/sec/ and 2 8-hours dark at 26±2 °C. Survival percentage was recordedat the end of three weeks. Acclimatized plants weretransferred to 12 x 23 cm polyethylene bags contain 1 soil:1 perlite mixture and grown under the greenhousecondition 24±2 °C day, 20±2 °C night and overheadirrigation. Statistical Analysis:  Each experiment was set up asacompletelyrandomized design. Collected data werestatistically analyzed using Statistical Analysis System of some Yemeni Coffea arabica  cultivars seedlings after 120 days (SAS) [14]. Means were separated according to the leastsignificant difference (LSD) test at 0.05 level of  probability. RESULTS AND DISCUSSION  In vitro germination:  Table 1 refers to the effect of different medium strength on the germination of thestudied coffee cultivars. It is clear that the totalgermination percentage decreased as the MS mediumstrength increased. However, agar medium only resultedhigher germination percentage for all studied cultivarsthan other MS medium strength. Lower germination percentage, resulted at high MS medium strength (fullstrength MS) could be due to high osmotic pressure of thegermination solution which makes imbibitions moredifficult and retards germination [14, 15]. Sterilization procedures resulted in low number of contaminatedcultures (Table 1) and these results are in agreement with previous result by Quirzo et al.  [16]. Furthermore, Table 2shows the effect of different medium strength on in vitro growth of coffee seedlings. After 120 days of inoculation,seeds (in the agar medium) germinated into plantlets withhigherroot length, more hypocotyl length and fullcotyledonary leaves expansion. While increased mediumstrength reduced the plantlets growthand cotyledonary.Maximum hypocotyl length 6.5-7.3 cm was obtainedon agar medium (Table 2). For all three cultivarssignificant differences were also obtained betweenmedia, as to root length. The maximum length of roots was9.8-11.6cm in the agar medium. There was not significant Table 1:Influence of different types of medium strength on germination of some Yemeni Coffea arabica  cultivarsMediumGermination %Contamination %OudayniAgar95121/4 MS 82121/2 MS 7814MS6618HammadyAgar98131/4 MS 86141/2 MS 6416MS5418DawaeiryAgar96101/4 MS 88101/2 MS 7212MS5813Table 2:Influence of different types of medium strength on in vitro growthof inoculationCotyledon leavesHypocotylRootSeedlingexpansion (+/-)Mediumlength (cm)length (cm)length (cm)OudayniAgar7.3 a9.8 a17.1 a+ y 1/4 MS 6.3 b9.2 a15.5 b-1/2 MS 2.7 c2.4 b5.1 c-MS1.5 d0.8 c2.3 d-HammadyAgar6.5 a10.8 a17.3 a+1/4 MS 6.1 b8.7 b14.8 b-1/2 MS 2.5 c2.2 c4.7 c-MS1.4 d0.7 d2.1 d-Dawaeiry Agar7.0 a11.6 a18.6 a+1/4 MS 6.7 b 10.3 b17 b-1/2 MS 2.8 c2.6 c5.4 c-MS1.6 d1.0 d2.6 d-y = Means within columns for the same cultivar having different letters aresignificantly different according to the least significant difference (LSD) at0.05 level of probability. (-) = Seedling without full cotyledon leavesexpansion. (+) = Seedling with full cotyledon leaves expansion difference between agar medium and quarter - strengthMS medium on root length of Oudayni seedlings whichresulted maximum seedling length of 17.1-18.6 cm in theagar medium. Thus, the differences obtained betweendifferent media strength were significant.  In vitro  shoot multiplication:  Table 3 shows the effectof BA concentrations in combination with 0.5 mg l IAA 1 on shoot multiplication of the studied coffee cultivars. Itis clear that after 120 days of culture, multiplication  World Appl. Sci. J., 2 (2): 142-150, 2007  145 Table 3:Influence of BA on number of shoots, shoot length, number ofTable 4:Influence of kinetin on number of shoots, shoot length, number leaves per explant and callusing of some in vitro  grown Yemeniof leaves per explant and callusing of some in vitro  grown Coffea arabica  cultivarsYemeni Coffea arabica  cultivarsBANumber ofShootNumber ofCallusingKinetinNumber ofShootNumber ofCallusingmg lshootslength (cm)leaves/explant(+/-)mg lshootslength (cm)leaves/explant(+/-) 1 OudayniOudayniC11.0 d1.4 bc4.3 d-C11.0 c1.4 b4.3 d- yz C21.0 d1.5 bc4.6 d-C21.0 c1.5 b4.6 d-2.04.1 c1.8 ab28.0 c-2.01.3 c1.8 ab9.4 c+4.07.7 b1.6 b36.0 bc+4.02.4 b1.7 b12.6 b+6.09.5 b1.4 ac41.8 b+6.03.0 b1.7 b13.3 b++8.016.8 a2.0 a89.0 a++8.05.2 a2.0 a29.2 a++HammadyHammadyC11.0 d1.6 b4.8 d-C11.0 c1.6 b4.8 d-C21.0 d1.6 b4.9 d-C21.0 c1.6 b4.9 d-2.03.7 c1.7ab27.0 c-2.01.2 c1.9 b8.8 c+4.07.1 b1.5 bc35.5 bc+4.02.2 b1.7 b11.8 bc+6.08.3 b1.5 bc38.3 b++bvv2.7 b1.6 b12.6 b+8.016.0 a2.1 a86.8 a++8.05.0 a2.4 a29.0 a++DawaeiryDawaeiryC11.0 d1.3 c4.0 d -C11.0 c1.3 c4.0 d-C21.0 d1.5 bc4.2 d-C21.0 c1.5 bc4.2 d-2.03.6 c1.7 ab26.3 c-2.01.1 c1.9 b8.6 c+4.06.5 b1.3 c34.2 bc+4.02.0 b1.8 b11.3 bc+6.07.7 b1.4 bc 37.4 b+6.02.4 b2.0 b12.3 b++8.015.4 a1.9 a85.1 a++8.04.4 a2.5 a27.9 a++y = C1 and C2 represent control treatments (with and without 0.5 mg ly = C1 and C2 represent control treatments (with and without 0.5 mg l 1 IAA, respectively). z = Means within columns for the same cultivar havingIAA, respectively). z = Means within columns for the same cultivar havingdifferent letters are significantly different according to the least significantdifferent letters are significantly different according to the least significantdifference (LSD) at 0.05 level of probability. (-) = no callus. (+, ++, +++,difference (LSD) at 0.05 level of probability. (-) = no callus, (+, ++, +++,++++, +++++, ++++++) = ( callus with 2-4, 4-6, 6-8, 8-10, 10-12,++++, +++++, ++++++) = (callus with 2-4, 4-6, 6-8, 8-10, 10-12,12-14, mm) diameter, respectively 12-14, mm) diameter, respectively  parameters and growth performance of these cultivarshighest number of leaves per ex-plant. The three studiedresponded significantly to increased BA concentrationscultivars exhibited similar response to BA concentrationsup to 8.0 mg l. This concentration (8.0 mg l BA)(Table 3). Result of Kahia and Owuor [17] showed 11 increased multiplication parameters significantly assuccessful in vitro  multiplication on solid MS mediacompared to other BA used concentrations. Shoot lengthsupplemented with 8.0 mg l BA combined with 0.8 mgand number of leaves produced from microshootsl IAA. Furthermore, Haidar [4] reported that thecultured on MS medium supplemented with 4.0 mg l BAcombination of 6-benzylaminopurine with 0.5 mg l IAA 1 was not significantly different from MS supplementedproduced the proliferation rate.with6.0 mg l BA in the three evaluated cultivars. In thisThe effect of different concentrations of kinetin 1 study,no significant differences appeared betweencombined with 0.5 mg IAA/l on in vitro  shootcontrol treatments, C1 (without 0.5 mg l IAA) andmultiplication of some Yemeni coffee cultivars is shown 1 C2 (with 0.5 mg l IAA) during 120 days of incubationin Table 4. It is obvious that the continuous increase in 1 and also no proliferation was occurred in both controls.kinetin concentrations to 8.0 mg l increased significantlyCallusing occurred at the basis of microshoots atthe shoots number (4.4-5.2), shoot height (2.0-2.5 cm) and4.0,6.0 and 8.0 mg BA/l. Results showed that theleaves number per ex-plant (27.4-29.2). Number of 9best concentration of BA which enabled C. arabica  proliferated shoots and leaves produced frommicroshoots to produce highest shoots number (15.4-16.8microshoots cultured on MS medium supplemented withshoots per explant) was 8.0 mg l combined with 0.5 mg4.0 mg l kinetin was not significantly different from 1 l IAA and resulted in highest shoot length andMS medium supplemented with 6.0 mg l kinetin in the 1 1yz1 111111  World Appl. Sci. J., 2 (2): 142-150, 2007  146 Table 5:Influence of TDZ on number of shoots, shoot length, number ofTable 6:Influence of 2ip on number of shoots, shoot length, number of leavesper explant and callusing of some in vitro  grown Yemenileaves per explant and callusing of some in vitro  grown Yemeni Coffea arabica  L. cultivars Coffea arabica  cultivarsTDZNumberShootNumber ofCallusing2ipNumberShootNumber ofCallusingmg lof shootslength (cm)leaves/explant(+/-)mg lof shootslength (cm)leaves/explant(+/-) 1 OudayniOudayniC11.0 c 1.4 a4.3 d-C11.01.4 b4.3 b- yz C21.0 c1.5 a4.6 d-C21.01.5 ab4.6 ab-2.01.7 b0.5 b5.4 b+++ ab5.2 ab+4.02.2 ab0.4 b6.0 ab++++ a5.4 ab+6.02.8 a0.3 b7.7 a+++++ a5.7 a+8.02.4 a0.2 b5.2 b++++++ ab4.3 b+HammadyHammadyC11.0 c1.6 a4.8 bc-C11.01.6 abc4.8 ab-C21.0 c1.6 a4.9 bc-C21.01.6 abc4.9 ab-2.02.0 b0.6 b5.8 bc+++ ab5.5 ab+4.02.4 b0.4 b6.2 b++++ a5.8 a+6.03.1 a0.3 b8.2 a+++++ abc5.1 ab+8.02.0 b0.3 b4.1 c++++++ c4.4 b+DawaeiryDawaeiryC11.0 b1.3 a4.0 bc -C11.01.3 b4.0 b-C21.0 b1.5 a4.2 bc-C21.01.5 ab4.2 b-2.01.3 b 0.5 b 4.6 bc+++ ab5.1 ab+4.01.6 b0.2 b5.3 b++++ a5.5 a+6.02.6 a0.4 b8.8 a+++++ a5.6 a+8.01.5 b0.2 b3.5 c++++++ ab4.0 b+y = C1 and C2 represent control treatments (with and without 0.5 mg ly = C1 and C2 represent control treatments (with and without 0.5 mg l 1 IAA, respectively). z = Means within columns for the same cultivar havingdifferent letters are significantly different according to the least significantdifference (LSD) at 0.05 level of probability. (-) = no callus. (+, ++, +++,++++, +++++, ++++++) = (callus with 2-4, 4-6, 6-8, 8-10, 10-12,12-14, mm) diameter, respectively studied Yemeni cultivars. It was reported thatmultiplication rate was increased in C. arabica  cv.Bourbon when MS medium was used and supplementedwith 15 mg l Kinetin [18]. 1 Allstudied levels of BA except control treatmentsshowed callus formation on the basal part of the proliferated shoots, the 6.0 and 8.0 mg l kinetin showed 1 higher callus formation in comparison to that of 2.0 and4.0 mg l kinetin. The effect of different concentrations 1 of TDZ combined with 0.5 mg l IAA on in vitro  shoot 1 multiplication of the three coffee cultivars is shown inTable(5). It is clear that 6.0 mg l TDZ caused the 1 highest significant increase in proliferated shoots andnumber of leaves/explant as compared to all other treatments.On the other hand the shoot length in thethree studied cultivars decreased with increasing TDZconcentration(Table 5). At concentration above 6.0 mgl TDZ, the number of proliferated shoots and the 1 number of leaves/explant significantly decreased. These 1yz1 IAA, respectively). z = Means within columns for the same cultivar havingdifferent letters are significantly different according to the least significantdifference(LSD) at 0.05 level of probability. (-) no callus. (+, ++, +++,++++, +++++, ++++++) = (callus with 2-4, 4-6, 6-8, 8-10, 10-12,12-14, mm) diameter, respectively results are in disagreement previous finding by Arafeh[19] who found that TDZ failed to promote proliferationin  Origanum  spp at the used levels. TDZ was a callusinducing factor and these results are in agreement withthe finding of Arafeh, [20], who reported that TDZ was acallus inducing factor in Origanum vulgare  L. The effectof different levels of 2ip and zeatin combined with 0.5 mgl IAA on in vitro  shoot multiplication of the studied 1 coffee cultivars are shown in Tables (6, 7). It is clear that both cytokinins failed to induced shoot multiplication atany of the used levels (2.0-8.0 mg l) and the growth of  1 mother stock (shoot length and number of leaves/explant)slightly increased in comparison to the control treatments.These results indicate that coffee did not responded tothe use of zeatin and 2ip, at least in the studied cultivarsat the used levels.  In vitro  rooting:  Microshoots of the three studiedcultivars were rooted on half-strength MS medium. The
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