Direct plant regeneration from cultured young leaf segments of sugarcane

Research note
Direct plant regeneration from cultured young leaf segments of sugarcane Raman Gill*,P.K.Malhotra&S.S.Gosal
Department of Plant Breeding,Genetics and Biotechnology,Punjab Agricultural University,Ludhiana,141 004,India(*requests for offprints:E-mail:rgillapril9@)
Received12April2005;accepted in revised form9August2005
Key words:hardened plantlets,organogenesis,profuse rooting,spindle explants,Saccharum officinarum L. Abstract
Young leaf segments(1.0–1.5cm)excised from spindle explants of three commercial sugarcane varieties viz. Co J64,Co J83and Co J86were cultured on different media compositions based on Murashige and Skoog (1962)salts.Cultured explants exhibited swelling followed by direct shoot regeneration on media containing naphthaleneacetic acid,in all the three varieties.Highest frequency83.12%shoot regeneration occurred on Murashige and Skoog medium supplemented with naphthaleneacetic acid(5.0mg l)1)and kinetin (0.5mg l)1)in variety Co J83.Medium devoid of naphthaleneacetic acid and supplemented with only kinetin did not induce direct shoot regeneration in any of the varieties thus tried.Subsequently profuse rooting of shoots was observed on the same medium and complete plantlets were recovered within6weeks. The plantlets were hardened and transferred to soil.Tissue culture derivedfield-grown plants were normal and exhibited faster growth and better tillering.This developed single step method of direct plant regen-eration can be used for rapid mass cloning and genetic transformation of sugarcane.
Abbreviations:2,4-D–dichlorophenoxyacetic acid;BA–benzylaminopurine;Kin–kinetin;MS–Murashige and Skoog;NAA–naphthaleneacetic acids
Sugarcane(Saccharum officinarum L.)is an important sugar crop possessing very high and variable chromosome number(2n=80)205).It is highly heterozygous and cross-pollinated in nature and requires very specific hot and humid climate forflowering.Therefore,the conventional breed-ing for incremental improvement is a difficult task. Besides,conventional propagation of sugarcane through sets is very slow,usually1–10in a year that is a major hurdle in the rapid spread of new varieties.Moreover,during vegetative propaga-tion,the pathogens keep on accumulating gener-ation after generation,which ultimately results in the decline of the variety.In this regard,the innovative approaches of cell and tissue culture and genetic transformation possess significant promise for creation of new genetic variability for improvement of sugarcane(Chahal and Gosal, 2002).Establishment of callus cultures(Cheema et al.,1992;Anbalogan et al.,2000),micropropa-gation through axillary shoot bud prolifera-tion(Gosal et al.,1998),somatic embryogenesis (Nieves et al.,1999;Gill et al.,2004),induction of somaclonal variation(Larkin and Scoworft,1983; Kaur et al.,2002)and stable genetic transforma-tion(Arencibia et al.,1998;Falcon et al.,2000) have been successfully achieved in sugarcane.In general,the use of callus based systems often cause genetic instability and lead to somaclonal varia-tion.Thus intervening callus phase is not desirable during micropropagation and genetic transforma-tion.Therefore in the present communication we report the development of a unique,efficient and highly reproducible single step method for
Plant Cell,Tissue and Organ Culture(2006)84:227–231ÓSpringer2005 DOI10.1007/s11240-005-9015-9
induction of direct plant regeneration from cul-tured young leaf segments of three commercial varieties of sugarcane.
Sugarcane tops measuring8–10cm were ex-cised from six-month-oldfield-grown plants of three commercial varieties viz.Co J64,Co J83 and Co J86of sugarcane.Outer whorls of mature leaves were removed till a spindle of5–6cm length and1.0cm diameter was obtained.Spindles were then surface sterilized using mercuric chloride (0.1%)for8min and washed thrice with sterile water.After removing outer two to three whorls of leaves,the innermost whorls of young leaves were cut into small segments(1.0–1.5cm)to serve as explants.These were then aseptically cultured on Murahige and Skoog(1962)solid medium supple-mented with various combinations and concentra-tions of naphthaleneacetic acid(NAA),kinetin (Kin)and benzylaminopurine(BA).Three hun-dred explants were taken per treatment and number of replications per treatment were three. The cultures were incubated under whiteflorescent light(68l mol m)2s)1),16/8h light/dark regimes at28°C.Data was subjected to statistical analysis as per Factorial Randomized Complete Block Design to see the level of significance and critical difference(CD)for media,variety and for variety Âmedia interaction.The obtained plantlets were washed thoroughly under running tap water to remove the media completely and hardened on water moist cotton in Enamel trays for1week under strongfluorescent light in the incubation room at28°C.Hardened plants were then trans-ferred to polythene bags containingfield soil and flyash(1:1)in the greenhouse.
The young leaf segments cultured on different media viz.M1,M2,M3,M4,M5and M6showed swelling and unwhorling during thefirst week of incubation in all the three varieties(Figure1a). Whereas,leaf segments cultured on media M7and M8supplemented with only Kin(0.5and 1.0mg l)1,respectively)remained quiescent and started turning brown after4weeks of incubation. During the third week of incubation,leaf segments cultured on auxin(NAA)and cytokinin(Kin and BA)containing media i.e.,M1,M2,M3,M4,M5 and M6exhibited direct shoot regeneration from their cut ends(Figure1b,c and d).It was interesting to note that maximum number of shoots that regenerated from one leaf segment were15–20in number.Explants of variety Co J83cultured on medium M1(MS+NAA 5.0mg l)1+Kin0.5mg l)1)showed highest (83.12%)shoot regeneration,that was followed by variety Co J64(77.61%)and variety Co J86 (74.37%)–(Figure2).With increase in the concentration of NAA from 5.0to10.0mg l)1 and Kin from0.5to1.0mg l)1in the media there was decrease in the percent shoot regeneration in all the varieties.The elevated concentration of NAA(10.0mg l)1)caused little callusing at the cut ends and thus reduced the percent shoot regener-ation in all the varieties.The leaf explants cultured on medium M3(MS+NAA10.0mg l)1+Kin
0.5mg l)1)exhibited reduced shoot regeneration
i.e.,78.08%in Co J83followed by Co J64
(68.00%)and Co J86(65.24%).Therefore addi-tion of auxin(NAA)was necessary for induction of direct shoot regeneration but increased concen-tration of NAA reduced the percent plant regen-eration.Addition of BA(1.0mg l)1)along with Kin and NAA in the medium i.e.,M5and M6did not show any synergistic effect in relation to direct shoot regeneration.Significant mean squares due to varieties and media revealed that varieties varied significantly from each other in response to direct plant regeneration.Different media tried were also significantly different with respect to direct plant regeneration.Variety mean squares and media mean squares were significant at5% level.With critical difference(CD)for media being 1.2,for variety0.85,CD for varietyÂmedia interaction at5%level of significance being2.09. Regenerated shoots in all the instances were normal,green and healthy.During thefifth week of incubation,profuse rooting(Figure1e)oc-curred from the base of plete separa-ble plantlets were recovered during the sixth week of incubation.Variety Co J83exhibited profuse rooting in comparison to Co J86and Co J64. Rooted shoot clumps when removed from the culture jars and transferred to Enamel trays continued to survive and did not wilt.During hardening of plantlets secondary and tertiary roots were seen to develop.Hardened plantlets trans-ferred to polythene bags in the greenhouse(Figure 1f)were healthy and green and95%survival was recorded.
Sugarcane breeding is a difficult task,therefore, the innovative cellular and molecular approaches are being sought for obtaining rapid incremental improvement in commercial varieties.Earlier
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reports on sugarcane particularly dealing with somatic embryogenesis and transformation are based on callus cultures.But excessive cell division during callus phase leads to somaclonal variation that is undesirable during cloning of elite clones and genetic transformation of commercial varie-ties.We have thus developed a unique direct plant regeneration method for cultured young leaf segments of three commercial varieties of sugar-cane.Although direct plant regeneration methods are available in dicots like tomato and potato (Webb et al.,1983;Asokan et al.,1984;Park et al.,1995)but similar systems are not available in monocots including sugarcane.In the present study shoots regenerated through organogenesis or direct somatic embryogenesis or both in the media containing both NAA and Kin.The roots developed later on the same medium suggest that high level of NAA more than 5.0mg l )1as not conducive for root formation,where as in the varieties lower concentration of NAA less than 5.0mg l )1was essential for rhizogenesis.No shoot regeneration was observed in medium M7and M8as it was devoid of auxin (NAA).Plant regenera-tion from variably old callus cultures,is well established in sugarcane (Heinz and Mee,1969;Ahloowalia and Meretzki,1983;Islam et al.,1996).But it is advantageous only when we are interested in obtaining somaclonal variations (Kaur et al.,2002)and is highly undesirable during conservation of germplasm in vitro ,mass cloning of newly developed varieties and during genetic transforma-tion for developing transgenic plants.Somatic embryogenesis which is being looked upon
as
Figure 1.(a )Cultured young leaf segments of sugarcane variety Co J 83showing swelling and unwhorling during first week of incubation.(b –c )Stereomicrograph (b –40Â,c –10Â)of direct shoot regeneration from cut ends of cultured young leaf segments in variety Co J 83on Murashige and Skoog medium supplemented with napthaleneacetic acid 5.0mg l )1and kinetin 0.5mg l )1.(d )Direct shoot regeneration on Murashige and Skoog medium supplemented with napthaleneacetic acid 5.0mg l )1and kinetin 0.5mg l )1in variety Co J 64.(e )Profuse rooting from base of shoots during the fifth week of incubation.(f )Tissue culture derived hardened plantlets of sugarcane variety Co J 83transferred to polythene bags in the greenhouse.(g )Direct shoot regenerated sugarcane plants of Co J 83in the field exhibiting normal growth and better tillering.
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an important aspect for cloning and genetic trans-formation has also been achieved in sugarcane.Various factors affecting induction,maintenance and regeneration of somatic embryos have been established (Fitch and Moore,1993;Aftab and Iqbal,1999).But these studies have followed the classical two step method i.e.,production of callus in the first step using high auxin (2,4-D )and low cytokinin (Kin or BA).In the second step shoot regeneration was obtained using medium contain-ing very low or no auxin and inclusion of cytoki-nins.The presence of even minimum callus interphase poses the problem of genetic instability.Therefore,in the present study 2,4-D that induces rapid callusing was replaced by a relatively weak auxin NAA.It was interesting to note that young leaf segments cultured on MS medium supple-mented with NAA (5.0mg l )1)and Kin (0.5mg l )1)did not exhibit any callusing and explant swelling was followed by direct shoot regeneration that is a rare phenomenon in mono-cots.The developed method is efficient,highly reproducible,is a single step method,and avoids callus interphase,therefore,it can be practically used for quick bulking of elite planting material of sugarcane.Besides this method can serve just like leaf disc method in dicots for efficient transforma-tion of sugarcane.References
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Figure 2.Percent direct plant regeneration from young leaf segments of three commercial sugarcane varieties cultured on different media compositions.M1–NAA (5.0mg l )1)+Kin (0.5mg l )1);M2–NAA (5.0mg l )1)+Kin (1.0mg l )1);M3–NAA (10.0mg l )1)+Kin (0.5mg l )1);M4–NAA (10.0mg l )1)+Kin (1.0mg l )1);M5–NAA (5.0mg l )1)+Kin (1.0mg l )1)+BA (1.0mg l )1);M6–NAA (10.0mg l )1)+Kin (1.0mg l )1)+BA (1.0mg l )1);M7–Kin (0.5mg l )1);M8–Kin (1.0mg l )1).
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