Development of an In Vitro Regeneration System in Sorghum [Sorghum bicolor (L.) Moench] Using Root Transverse Thin Cell Layers (ttcls)

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1 Turk J Bot 3 (26) 1-9 TÜB TAK Researh Artile evelopment of an In Vitro Regeneration System in Sorghum [Sorghum iolor (L.) Moenh] Using Root Transverse Thin Cell Layers (ttcls) P. BASKARAN, B. RAJA RAJSWARI, N. JAYABALAN Plant Biotehnology Unit, epartment of Plant Siene, Shool of Life Sienes, Bharathidasan University, Tiruhirappalli 62 24, Tamil Nadu, INIA Reeived: Aepted: Astrat: An effiient and reproduile plant regeneration system was developed from ells or tissues of agronomially important Indian sorghum genotypes inluding 2 ommerial ultivars (NS27 & K8) of Sorghum iolor (L.) Moenh. Callus indution and plant regeneration were ahieved on transverse thin ell layers (ttcl) of roots from aseptially germinated 7-day-old seedlings. Callus response depended on the genotype, the onentrations and omposition of growth sustanes and numer of in vitro regeneration yles undergone y the donor plant. Murashige and Skoog (MS) medium supplemented with µm 2,4- dihlorophenoxy aeti aid (2,4-), µm naphthalene aeti aid (NAA), µm indole aeti aid (IAA) and µm indole utyri aid (IBA), and omined with 1% (v/v) oonut water (CW) was used for allus indution. The alli were ultured on MS medium supplemented with µm 6-enzylaminopurine (BAP) omined with 2.3 µm 2,4- or 2.7 µm NAA. ighly effiient differentiations of multiple shoot uds were initiated within 4 weeks of ulture. Root indution was ahieved on halfstrength MS medium ontaining IAA ( µm). Rooted plants were suessfully alimatised, with the survival rate reahing almost 8%. These plants grew normally without showing any morphologial variation. Key Words: Callus indution, oonut water, hardened plant, MS medium, plant growth regulators, regeneration, rooting Introdution Sorghum Moenh is an important ereal rop oupying a major plae in oth food grain and forage prodution. The rop is well adapted to tropial and sutropial areas throughout the world. In addition to its priniple uses as flour, in the preparation of porridge and unleavened read, Sorghum speies are soures of fire, fuel and seondary produts and are also used in the alohol industry (sweet Sorghum) as they ontain high amounts of starh. In the developing world, improving sorghum through iotehnology is the latest in a long series of tehnologies that have een applied to this rop (Maqool et al., 21). owever, transverse thin ell layer (ttcl) is a model system whih has appliations in higher plant tissue and organ ulture, and geneti transformation. Sine the regeneration of speifi organs may e effetively manipulated through the use of ttcls, in onjuntion with speifially ontrolled in vitro onditions and exogenously applied plant growth regulators (PRs), many prolems hindering the improvement of in vitro plant systems are potentially removed (Jaime & Teixeira da Silva, 23). TCL tehnology has een effetively used in ereals and grasses, inluding igitaria sanguinalis (L.) Sop., Oryza sativa L. and Zea mays L. The transverse ttcl tehnology has een suessful in many plant speies (Pelissier, 199; Ohki, 1994; Stefaniak, 1994; osokawa et al., 1996; Bui et al., 1998a; Baskaran and Jayaalan, 25). Plant regeneration in sorghum has een desried using various explants (amorg et al., 1977; Thomas et al., 1977; Ma et al., 1987; Zhong et al., 1988; agio, 22; Mishra and Khurana, 23). Cereal tissue ultures produe different types of alli (i.e. masses of undifferentiated ells), whih may differ in their regenerative potentials. Most ereals seem to produe allus tissue with and without green spots, and a positive orrelation etween the presene of suh spots in a allus and its regenerative potential has een oserved (Ogura & Shimada, 1978; Shimada & Yamada, 1979; Inoue & Maeda, 198; Naors et al., 1982). Suessful ulturing of allus has een reported for orn (Masarenhas et al., 1965), oats (Carter et al., 1

2 evelopment of an In Vitro Regeneration System in Sorghum [Sorghum iolor (L.) Moenh] Using Root Transverse Thin Cell Layers (ttcls) 1967), rie (Yatazwa et al., 1967) and wheat (Troine et al., 1968). In sorghum, immature infloresene was used as potential explants for regeneration (lkonin et al., 1996; Raghavendra Rao et al., 2). Sine an effiient and reproduile regeneration protool is required efore any geneti transformation study, in the present ommuniation, the regeneration ailities of Sorghum iolor vs. NS27 and K8 were examined. The purpose of the investigation reported here is to provide a simple, reproduile and effiient in vitro ulture system for sorghum (Sorghum iolor). Materials and Methods Plant material Seeds of Sorghum iolor vs. NS27 and K8 were otained from Tamil Nadu Seed ermination Testing Laoratory, Tiruhirappalli, India. The seeds were kept under running tap water for 1 h efore eing washed with an aqueous solution of 2% (v/v) Teepol (Rekitt Benkiser, India) for 3 min, followed y rinsing with distilled water and 7% (v/v) ethanol for 1 min with further 3 to 5 rinsings in sterile distilled water. The seeds were then surfae sterilised with.2% (w/v) aqueous meruri hloride solution for 1 min and finally rinsed with sterile distilled water (5 to 7 hanges). The seeds were then germinated on autolaved MS asal medium or in moistened otton. The root segments derived from 7- day-old asepti seedlings were used as explants. The root segments (.3-.5 mm) were disseted transversely in asepti onditions. Culture onditions Murashige and Skoog (1962) medium (MS) supplemented with 3% (w/v) surose (imedia, India) was used in all the experiments. The p of the medium (supplemented with respetive growth regulators) was adjusted to 5.8 with 1 N NaO or 1 N Cl prior to the addition of.8% (w/v) agar. In all the experiments, the hemials used were of analytial grade (imedia, Qualigens, Merk, Loa Chemie, isher and Sigma). The medium was dispensed into ulture vessels (Borosil, India) and plugged tightly with non-asorent otton and autolaved at 15 kpa (121 C) for 15 min. All the ultures were maintained at 25 ± 2 C under a 16-h photoperiod of 45-5 mmol m -2 s -1 irradiane provided y ool white fluoresent tues (Philips, India) and with 55%-6% relative humidity (R). All susequent suultures were performed at 4-week intervals. Callus indution medium Transverse TCL segments (.3-.5 mm) of roots were ultured on MS medium supplemented with 1% (v/v) oonut water (CW), and with different onentrations and ominations of PRs, inluding µm 2,4-, µm NAA, µm IAA and µm IBA. Plant regeneration medium White friale alli (3-days-old) were ultured on MS medium supplemented with 5% oonut water (CW) and different onentrations and ominations of PRs, inluding µm BAP and the addition of 2.3 µm 2,4-. Rooting medium longated shoots were exised from eah ulture passage and transferred to half-strength MS medium (1/2 MS) supplemented with different onentrations of IAA ( µm). Alimatisation and transfer of plantlets to soil Plantlets with well-developed roots were removed from the ulture medium, the roots were washed gently under running tap water, and they were transferred to plasti pots (1 m diameter) ontaining a mixture of autolaved garden soil, farmyard soil and sand (2:1:1), respetively. All pots were irrigated with 1/8 MS asal salt solution devoid of surose and inositol every 4 days for 2 weeks. The potted plantlets were overed with porous polyethylene sheets for maintaining high humidity and were maintained under the ulture room onditions. The relative humidity was redued gradually. After 3 days, the plantlets were transplanted to a otanial evaluation garden and kept under shade in a net house for further growth and development. Statistial analysis xperiments were set up in a randomised lok design (RB), with 3 repliations. Ten to fifteen explants were used per treatment in eah repliation. Oservations were reorded on the perentage of response of allus formation, perentage of response of shoots, numer of shoots per allus, shoot length, perentage of response of roots, roots per shoot and root length. The treatment means were ompared using unan s multiple range test (MRT) at a 5% proaility level aording to omez and omez (1976). 2

3 P. BASKARAN, B. RAJA RAJSWARI, N. JAYABALAN Results and isussion Callus Indution Cv. NS27 was more responsive to media for allus indution and shoot regeneration ompared with K8 (igure 1a & ). Callus initiation was oserved in NS27 within 1 days, ut in K8 after 15 days of ulture. Optimum allus prodution was oserved in NS27 after 3 days of ulture, whereas lower allus growth was oserved in K8. requeny of allus indution, type of allus and regeneration of plantlets were influened y the genotypes. A similar phenomenon was reported y Patil et al. (1998). The allus ultured for plant regeneration medium after the first suulture was very effetive in otaining high regeneration potential. The seond and third suulture yles resulted in the rowning of alli. Initially, yellowish ompat allus was formed diretly at ttcl of roots ontaining MS medium fortified with auxins, ut this allus later turned whitish ompat after 3 days of ulture in oth ultivars. owever, most of the ultivars formed yellowish ompat allus as desried y agio (1994). Among the 4 auxins, only 2,4- was effetive for allus growth in oth ultivars. Similar results were also oserved y Arti et al. (1994), Nguyen et al. (1998), Saradamani et al. (23) and Baskaran and Jayaalan (25). Callus growth of S. iolor was highly influened y onentrations of growth regulators and oonut water (CW) added to the ulture medium. Addition of CW [1% (v/v)] to MS medium fortified with 2,4- inreased allus growth in v. NS27 (igure 5A). A similar result was oserved in sugarane (Mamun et al., 24). Among the 4 auxins, 2,4- (9. 5VSRQV ' &: 1$$ &:,$$ &:,%$ &: 3OQW JURZWK UJXOWRUV 5VSRQV ' &: 1$$ &:,$$ &:,%$ &: 3OQW JURZWK UJXOWRUV igure 1. ffet of different onentrations of auxins with 1% CW on the mean perentage of allus prodution (a v NS27 and K8). The ars earing a mean followed y different letters on top are signifiantly different from eah other (P <.5); omparison y MRT. ata reorded after 3 days of ulture. 3

4 evelopment of an In Vitro Regeneration System in Sorghum [Sorghum iolor (L.) Moenh] Using Root Transverse Thin Cell Layers (ttcls) µm) with 1% CW proved to e etter for allus growth than the other PRs. A similar result has een reported for the ttcl hypootyl explant in S. iolor (Baskaran and Jayaalan, 25). On the other hand, allus an also e grown on medium without CW ut at a muh slower rate in oth ultivars (data not shown) as in line with Vane and avid (197). The ranges of µm 2,4-, µm NAA, µm IAA and µm IBA with 1% CW were found to e optimal for otaining white friale allus after 3 days of ulture (igure 1a & ). owever, the medium ontaining µm NAA or µm IAA or µm IBA and 1% CW produed roots on surfae of the white ompat allus. This ourred vigorously in v. K8 after 45 days of ulture. Shoot Regeneration xperiments were designed to improve plant regeneration from allus derived from roots (ttcl) in Sorghum iolor (ultivar NS27 and K8). Indution of organogeni allus and shoot regeneration ourred on MS medium supplemented with 5% CW and different onentrations of BAP ( µm), and omined with 2,4- (2.3 µm) or NAA (2.7 µm). ttcl root allus produed shoot regeneration in NS27 (igure 2). Teixeira da Silva (23) and Teixeira da Silva and ukai Response (%) a a a a a d de d BAP+2,4- (2.3) BAP+NAA (2.7) Plant growth regulators (µm) Numer of shoots per allus BAP+2,4- (2.3) BAP+NAA (2.7) Conentrations of BAP (µm) Mean shoot length (m) per ulture BAP+2,4- (2.3 BAP+NAA (2.7) Conentrations of BAP (µm) igure 2. ffet of different onentrations of BAP with 5% CW and addition of auxins on the mean perentage of shoots response (a), the mean shoot numer (), the mean shoot length (). The ars earing a mean followed y different letters on top are signifiantly different from eah other (P <.5); omparison y MRT. rror ars represent the standard error. ata reorded after 8 weeks ulture of S. iolor (v. NS27). 4

5 P. BASKARAN, B. RAJA RAJSWARI, N. JAYABALAN (23) reported that ttcls were effetive in shoot regeneration and morphogenesis in Chrysanthemum L. owever, higher onentrations of 2,4- (aove 2.3 µm) and NAA (aove 2.7 µm) in the regeneration medium resulted in dereased frequenies of shoot regeneration (data not shown) in oth ultivars. These results were in agreement with Bhaskaran et al. (1992) and Murray et al. (1983). igher levels of 2,4- (aove 13.6 µm) in allus prodution medium slowed down the susequent plant formation on regeneration medium. A similar phenomenon was oserved y Murray et al. (1983). The synergisti effet of MS medium ontaining BAP at and 17.8 µm, 2,4- (2.3 µm) and 5% CW was found to e optimum for shoot initiation (igure 5B). The maximum numer of shoots was oserved in NS27 after 4 weeks (igure 2; igure 5C). In oth ultivars, the maximum numer of shoots was otained in MS medium ontaining BAP (13.3 µm), 2,4- (2.3 µm) and 5% CW ut shoot lengths varied after 8 weeks (igure 2; igure 5). igher onentrations of BAP (aove 13.3 µm) redued the perentage of response, numer of shoots and shoot lengths. Culturing the alli on the medium supplemented with BAP ( µm), NAA (2.7 µm) and 5% CW resulted in shoot indution in oth ultivars. A marked response as well as shoots regeneration was otained in medium ontaining BAP Rresponse (%) Numer of shoots per allus d a BAP + 2,4- (2.3) BAP + NAA (2.7) Plant growth regulators (µm) Conentrations of BAP (µm) de d a BAP+2,4- (2.3) BAP+NAA (2.7) a Mean shoot length per ulture (m) BAP+2,4- (2.3) BAP+NAA (2.7) Conentrations of BAP (µm) igure 3. ffet of different onentrations of BAP with 5% CW and addition of auxins on the mean perentage of shoots response (a), the mean shoot numer (), the mean shoot length (). The ars earing a mean followed y different letters on top are signifiantly different from eah other (P <.5); omparison y MRT. rror ars represent the standard error. ata reorded after 8 weeks ulture of S. iolor (K8). 5

6 evelopment of an In Vitro Regeneration System in Sorghum [Sorghum iolor (L.) Moenh] Using Root Transverse Thin Cell Layers (ttcls),q YLWUR URRW IRUPWLRQ UVSRQV URRW QXPU URRW OQJWK &RQQWUWLRQV RI,$$ igure 4. ffet of half-strength MS medium with different onentrations of IAA on the Mean perentage of roots response ( ), the mean root numer ( ), the mean root length (m) ( ). The line earing mean followed y different letters in their top are signifiantly different from eah other (P <.5); omparison y MRT. rror ars represent the standard error. ata reorded after 3 days ulture of S. iolor (NS27). (13.3 µm), NAA (2.7 µm) and 5% CW in NS27 ut with variale shoot lengths (igure 2 & ). Auahker and Murugesan (1999) have reported the onentration of BAP (6.6 µm) and NAA (2.7 µm) to e the most ongenial for shoot regeneration in young stem explants of S. iolor. owever, Nirwan and Kothari (24) have reported BAP (8.8 µm) and IAA (2.9 µm) to e the effetive for shoot ud regeneration in apial meristem explants in S. iolor. On the other hand, shoot regeneration ourred on MS medium ontaining kinetin (9.2 µm) and IAA (2.85 µm) in mature emryo allus (Nirwan and Kothari, 23). In this study, we oserved the ulture response as well as shoot numer and shoot length suordinate in K8 (igure 3a, & ). Rooting of Shoots xised shoots from oth ultivars of Sorghum iolor were rooted on half-strength MS medium with IAA ( µm). No signifiant differenes were oserved etween the ultivars with respet to rooting (data not shown). The promotory effet of reduing the salt onentration of MS on in vitro rooting of shoots has een desried in several reports (Constantine, 1978; Skirvin et al., 198). alf-strength MS medium supplemented with all onentrations of IAA indued roots from shoots within 15 days of ulture. Among the IAA onentrations, the perentage of response, numer of roots and root length varied (igure 4). alf-strength MS medium supplemented with IAA (22.8 µm) was the most effetive for root indution (igure 4; igure 5). Sarada Mani et al. (23) reported that MS medium supplemented with IAA produed roots. owever, Nirwan and Kothari (24) reported half-strength MS medium ontaining 1.7 µm NAA and 2% (w/v) surose to e the most ongenial for root indution. Alimatisation and ield stalishment Rooted plantlets were suessfully alimatised without the need for a growth hamer faility. One hundred perent of the plantlet survival was seen after hardening on garden soil, farmyard soil and sand (2:1:1) for 3 weeks. ardened plantlets were suessfully transferred to a otanial evaluation garden and kept under shade in a net house for further growth and development after 3 weeks (igure 5). owever, the survival rate dereased from 1% to 8% after 1 weeks of alimatisation. There was no variation among the alimatised plants omparale to in vivo plants with respet to morphologial, growth haraters and yield. All the in vitro derived plantlets were free from external defets. Conlusion The purpose of this study was to develop an in vitro propagation method on ttcl tehnology of root segments in S. iolor, an eonomially important rop plant. Various plant growth regulators and oonut water 6

7 P. BASKARAN, B. RAJA RAJSWARI, N. JAYABALAN igure 5. A-. Organogenesis from roots (ttcl) of Sorghum iolor (v. NS27). (A) Indution of white friale allus from ttcl (root). (B) Shoot ud initiation on MS medium with p13.3 µm BAP and 2.3 µm 2,4-. (C) Regenerated shoots after 3 days of ulture. () longated multiple shoots after 5 days of ulture. () Rooted plantlet of S. iolor. () Alimatised plantlets of S. iolor. 7

8 evelopment of an In Vitro Regeneration System in Sorghum [Sorghum iolor (L.) Moenh] Using Root Transverse Thin Cell Layers (ttcls) were tested for a rapid and reproduile method of shoot proliferation using ttcl from root segments, whih was followed y suessive estalishment of regenerated plants in soil. The protool reported here ould e used for large-sale propagation of this valuale rop plant. The ttcl in vitro system desried here provides an effiient regeneration protool for Sorghum iolor. Aknowledgements The first author is grateful to r. V.T. Sridharan, Rev. r. S. John Britto and r. Patrik omez for their ritial appraisal of the paper. Many thanks are due to A. Bakrudeen and. Sujatha for their valuale help during the preparation of the manusript. The author wishes to thank.. ideon for the helpful disussion and ritial reading of the manusript. Referenes Auahker MN & Murugesan NP (1999). In vitro organogenesis of various explants of Sorghum iolor (L.) Moenh (Sweet sorghum). Plant Tiss Cult 9: Arti P, evi R & Murty VR (1994). mryogeni allus indution and plant regeneration in forage Sorghum. Cereal Res Com 22: Baskaran P & Jayaalan N. (25). A simple approah to improve plant regeneration from allus ulture of Sorghum iolor for rop improvement. J Agri Bioteh 1: Bhaskaran S, Rigoldi M & Smith R (1992). evelopmental potential of Sorghum shoot apies in ulture. Journal of Plant Physiology 14: Bui VL, Nghieng Thao M, Jeanneau M, Sadik S, Tu S, Vidal J & Tran thanh Van K (1998a). Rapid plant regeneration of a C 4 diot speies: Amaranthus edulis. Plant Si 132: Carter O, Yamada Y & Takahashi (1967). Tissue ulture of oats. Nature 214: Constantine (1978). Round Tale Conferene, emloux, Belgium. pp 134. lkonin LA, Lopushanskaya R & Pakhomova NV (1996). mryogeni allus of Sorghum (Sorghum iolor (L.) Moenh) y amino aids. Maydia 4: amorg OL, Shyluk JP, Brar S & Constale (1977). Morphogenesis and plant regeneration from allus of immature emryos of sorghum. Plant Si Lett 1: omez KA & omez AA (1976). Statistial proedures for agriultural researh with emphasis on rie. Los Banos, Philippines: International Rie Researh Institute. agio T (1994). Varietal differene of plant regeneration from allus of sorghum mature seed. Breeding Siene 44: agio T (22). Adventitious shoot regeneration from immature emryos of sorghum. Plant Cell Tiss Org Cult 68: osokawa K, Nakano M, Oikawa Y & Yamamura S (1996). Adventitious shoot regeneration from leaf, stem and root explants of ommerial ultivars entiana. Plant Cell Rep 15: Inoue M & Maeda (198). ffets of auxins and ytokinins on the ourrene of green regions in rie allus ultures. Jpn J Crop Si 49: Jaime A & Teixeira da Silva (23). Thin Cell Layer tehnology in ornamental plant miropropagation and iotehnology. Afrian Journal of Biotehnology 2: Ma, U M & Liang. (1987). Plant regeneration from ultured immature emryos of Sorghum iolor (L.) Moenh. Theor App enet 73: Mamun MA, Sikdar MB, ipak Kumar Paul, Mizanur Rahman & Rezuanul Islam (24). In vitro miropropagation of some important sugarane varieties of Bangladesh. Asian Journal of Plant Sienes 3: Maqool SB, evi P, Stiklen MB (21). Biotehnology: eneti improvement of sorghum (Sorghum iolor (L.) Moenh). In Vitro Cell ev Biol Plant 37: Masarenhas A, Sayagaver BM & Jagannathan V (1965). Studies on the growth of allus ultures of Zea mays. In: Ramkrishnan v (ed.). Tissue Culture seminar Proeedings, University Of Baroda, India, The ague (Netherlands): W. Junk Pul. pp Masteller VJ & olden J (197). The growth of and organ formation from allus tissue of Sorghum. Plant Physiol 45: Mishra A, Khurana P (23). enotype dependent somati emryogenesis and regeneration from leaf ase ultures of Sorghum iolor. J of Plant Biohem Bioteh 12: Murray WN, James W, Thomas A & Kiry J (1983). Long-duration, high frequeny plant regeneration from ereal tissue ultures. Planta 157: Naors MW, Kroskey CS & Mugh M (1982). reen spots are preditors of high allus growth rates and shoot formation in normal and in salt stressed tissue ultures of oat (Avena sativa L.) Z Pflanzenphysiol 15: Nguyen TV, odwin I, Ale JA, ray SJ & Rathus C (1998). ffet of plant growth regulators on emryogeni allus formation and growth in vitro of grain Sorghum. International Sorghum and Millets News letter (India). 39: ordreht, the Netherlands. Nirwan RS & Kothari SL (23). igh opper levels improve allus indution and plant regeneration in Sorghum iolor (L.) Moenh. In Vitro Cell ev Biol Plant 39:

9 P. BASKARAN, B. RAJA RAJSWARI, N. JAYABALAN Nirwan RS, Kothari SL (24). igh frequeny shoot organogenesis in Sorghum iolor (L.) Moenh. J of Plant Biohem Bioteh 13: Ogura & Shimada T (1978). Preliminary report on shoot redifferentiation from wheat allus. Wheat Inf Serv 45: Ohki S (1994). Sanning eletron mirosopy of shoot differentiation in vitro from leaf explants of the Afrian violet. Plant Cell Tiss Org Cult 36: Patil Vishwanath M & Kuruvinashetti MS (1998). Plant regeneration from leaf sheath ultures of some rai sorghum ultivars. South Afrian Journal of Botany 64 (3): Pelissier B, Bouhefra O, Pepin R & reyssinet (199). Prodution of isolated Somati emryos from sunflower thin ell layers. Plant Cell Rep 9: Raghavendra Rao RV, Pavan Kumar, Sua Rao MV & Manga V (2). ifferential in vitro response of genotypes of Sorghum. Phytomorph 5: Saradamani N, Muralimohan S, Sudhakar RP & ora (23). In vitro morphogenesis in ultivated varieties of Sorghum iolor (L.) Moenh. Plant Cell Biotehnology and Moleular Biology 4: Shimada T & Yamada Y (1979). Wheat plants regenerated from emryo ell ultures. Jpn J enet 54: Skirvin RM, Chu MC & Rukan (198). An improved medium for the in vitro rooting of arrite peah. Pro III State orti So113: Stefaniak B (1994). Somati emryogenesis and plant regeneration of ladiolus. Plant Cell Rep 13: Teixeira da Silva JA (23). Tissue ulture and ryopreservation of hrysanthemum: a review. Biotehnol Adv 21: Teixeira da Silva JA & ukai S (23). Chrysanthemum organogenesis through thin ell layer tehnology and plant growth regulator ontrol. Asian J Plant Si 2: Thomas, King PJ & Portykus I (1977). Shoot and emryo-like struture formation from ultured tissues of Sorghum iolor. Naturwissenshaften 64: 587. Trione J, Jones L & Metzger RJ (1968). In vitro ulture of somati wheat allus tissue. Amer J Bot 55: Yatazwa M, uruhashi K & Shimizu M (1967). rowth of allus tissue from rie root in vitro. Plant Cell Physiol 8: Zhong, Wang W & Stiklen M (1998). In vitro regeneration of Sorghum iolor (L.) Moenh: effiient plant regeneration from shoot apies. J Plant Physiol 153: